JP6605435B2 - Method for modifying isoelectric point of antibody by amino acid substitution of CDR - Google Patents

Description

The present invention relates to a method for modifying the isoelectric point while maintaining the binding activity of an antibody to an antigen, a method for controlling the plasma pharmacokinetics (blood kinetics) of an antibody, and the isoelectric point being modified by amino acid substitution of CDR. The present invention relates to a pharmaceutical composition containing the prepared antibody as an active ingredient, a method for producing the same, and the like.
The present invention also provides an anti-IL-6 receptor antibody, an anti-glypican 3 antibody, and an anti-IL-6 receptor antibody, an anti-IL-6 receptor antibody, an anti-IL-6 receptor antibody, an anti-IL-6 receptor antibody, Methods for controlling plasma half-life of anti-IL-31 receptor antibody and anti-IL-31 receptor antibody, antibodies whose plasma half-life is controlled by modification of amino acid residues (anti-IL-6 receptor antibody, anti-glypican 3 antibody, and The present invention relates to an anti-IL-31 receptor antibody), a pharmaceutical composition containing the antibody as an active ingredient, and a method for producing the pharmaceutical composition.
Furthermore, the present invention relates to a pharmaceutical composition containing an anti-IL-6 receptor antibody as an active ingredient, a method for producing the same, and the like.

  Antibodies are attracting attention as pharmaceuticals because of their long plasma half-life and few side effects. Among them, many IgG-type antibody drugs are on the market, and many antibody drugs have been developed (Non-patent Documents 1 and 2). Currently, most antibody drugs on the market are chimeric antibodies, humanized antibodies, and human antibodies. However, humanized antibodies or human antibodies have been improved to improve drug efficacy, convenience, and cost, and have superior characteristics. Many medicines are currently being developed. Various technologies have been developed that can be applied to these antibody drugs, and technologies that improve effector function, antigen binding ability, pharmacokinetics, stability, or reduce immunogenicity risk have been reported. Yes. As a method of enhancing the drug efficacy or reducing the dose, there is a technique for enhancing antibody-dependent cytotoxic activity (ADCC activity) or complement-dependent cytotoxic activity (CDC activity) by amino acid substitution in the Fc region of IgG antibody. It has been reported (Non-Patent Documents 3 and 4). In addition, affinity maturation technology (Non-Patent Document 5) has been reported as a technology for improving antigen binding ability and antigen neutralization ability, and by introducing mutations into amino acids such as CDR regions of variable regions, It is possible to improve the binding activity.

  A problem with current antibody drugs is high production cost due to the very large amount of protein administered. As for the form of administration, in the case of chronic autoimmune disease, a subcutaneous preparation is desirable, but in general, the subcutaneous preparation needs to be a high concentration preparation. In the case of an IgG type antibody preparation, stability, etc. In general, it is considered that the limit is about 100 mg / mL (Non-patent Document 6). Prolonged plasma half-life of the antibody to reduce the amount of protein to be administered so that sustained therapeutic effects can be achieved, enabling subcutaneous administration at long dosing intervals, and has excellent properties that are low in cost and convenient. It is possible to provide antibody drugs.

  FcRn plays a major role in the long plasma half-life of antibodies, with regard to differences in plasma half-life between antibody isotypes, IgG1 and IgG2 have the longest plasma half-life and IgG3 and IgG4 are inferior It is known (Non-Patent Document 7). As a method for further extending the plasma half-life of IgG1 and IgG2 antibodies having excellent plasma half-life, amino acid substitution in the constant region that enhances binding to FcRn has been reported (Non-patent Documents 8, 9, and 10). . However, the introduction of artificial amino acid mutations into the constant region has a problem from the viewpoint of immunogenicity. In contrast, a method for improving the pharmacokinetics of an antibody by introducing a mutation into an amino acid in the variable region of the antibody has recently been reported (Patent Document 1).

  According to Patent Document 1, it is possible to control the pharmacokinetics of IgG by changing the isoelectric point, and to reduce the binding activity of the antibody to the antigen by introducing an amino acid substitution into the framework of the antibody variable region It has been reported that the isoelectric point of an antibody can be decreased without increasing the plasma half-life of the antibody. Specifically, for example, by introducing amino acid substitutions in H10, H12, H23, H39, H43, and H105 in Kabat numbering, the isoelectric point of the antibody can be lowered without diminishing the binding activity of the antibody to the antigen. Is possible. In addition, amino acid mutations can be introduced into other framework sequences without attenuating the binding activity, but in order to significantly reduce the isoelectric point, introduction of amino acid substitutions into the framework sequence is possible. In some cases, it was not enough. This is because framework sequences after amino acid substitution generally use human antibody sequences to make them less immunogenic, but human antibody framework sequences are highly conserved and less diversified, so they are free from amino acid substitutions. This is because the degree is small. Therefore, when it is insufficient to reduce the isoelectric point of an antibody only by introducing an amino acid substitution into the framework, it is difficult to further reduce the isoelectric point.

  On the other hand, CDR sequences have enormous diversity due to somatic mutation and diversity to acquire binding to antigens, so the degree of freedom of amino acid substitution is significantly higher than that of the framework. large. However, the CDR sequence is the most important element for exerting a strong binding activity to the antigen, and it is generally known that amino acid substitution of the CDR sequence affects the binding activity of the antibody to the antigen. For this reason, it is difficult to reduce the isoelectric point of an antibody without significantly reducing the binding activity of the antibody to the antigen by amino acid substitution of the CDR sequence. In addition, since the CDR sequence varies greatly depending on the type of antigen, it does not depend on the type of antibody, and it is extremely difficult to replace the amino acid of the CDR sequence of the antibody without significantly reducing the binding activity of the antibody to the antigen. It has been thought that there is. In fact, this can be easily inferred from the many events shown below.

  When humanizing an antibody of a non-human animal species, CDR grafting in which a CDR sequence of the non-human animal species is transplanted to a human framework sequence is generally used. If the humanized antibody obtained by CDR grafting does not show the same binding activity as the chimeric antibody, a part of the framework sequence that determines the structure of CDR is part of the antibody framework of the non-human animal species from which the antibody is derived. Binding activity can be recovered by amino acid substitution in the sequence (Non-patent Document 11). Thus, the CDR sequence and structure are extremely important for the antigen-binding activity and specificity of the antibody. In addition, aspartic acid isomerization reaction, asparagine residue deamidation reaction, and antibody CDR residue change due to oxidation reaction of methionine residue in antibody CDR are widely known. Therefore, the CDR sequence is extremely important for the binding activity of the antibody to the antigen. Furthermore, when amino acid substitutions are introduced into the H chain CDR2 sequence of an antibody, it has been reported that in many cases, the antigen binding activity is greatly reduced and the expression level of the antibody is also reduced (Non-patent Documents 13 to 15). . In particular, it has been found that when an amino acid substitution is introduced into H51, the expression level of the antibody is remarkably reduced (Non-patent Document 16). In addition, it has been reported that when a mutation is introduced into the H chain CDR3 sequence of an antibody, in most cases, the antigen binding activity is greatly reduced (Non-patent Documents 17 and 18). In addition, when alanine scanning of the CDR sequence of an antibody is performed, in many cases, the binding activity to the antigen of the antibody is greatly reduced by substituting the amino acid present in the CDR with alanine (Non-patent Document 19). -23), and the influence on the binding activity to the antigen when substituted with alanine is considered to vary depending on the type of antibody. In other words, it is generally considered that the binding activity to the antigen is attenuated by amino acid substitution of the CDR sequence of the antibody, and the amino acid substitution site that does not greatly reduce the binding activity of the antibody to the antigen regardless of the type of antibody. There is no report so far.

  In antibody engineering for creating antibody molecules having superior properties, amino acid substitutions to antibody CDR sequences are mostly performed for the purpose of affinity maturation. Affinity maturation generally displays antibody libraries with randomized CDR sequences on the phage or ribosome against the CDR sequence of an antibody molecule, and further improves the binding activity to the antigen by panning to the antigen. In this method, it is possible to find amino acid substitutions in the CDR sequence of an antibody that improves the binding activity to the antigen (Non-patent Documents 5, 24 to 26). However, since the amino acid substitution that improves the binding activity to the antigen obtained by this method differs depending on the type of antibody, the amino acid substitution site in the CDR sequence that improves the binding activity to the antigen does not depend on the type of antibody. There is no report until. Other than affinity maturation, a method for improving the expression level of an antibody in a mammalian cell by substituting an amino acid of a CDR sequence at a specific position has been reported (Patent Document 2). According to Patent Document 2, it is possible to improve the expression level of an antibody in mammalian cells without depending on the type of antibody, by substituting a specific sequence for the amino acid of a CDR sequence at a specific location. In addition, de-immunization that avoids T-cell epitopes present in the CDR sequence of antibodies to reduce the immunogenicity of antibodies has been reported. No method for amino acid substitution that removes the T-cell epitope present in the CDR sequence without attenuation has been reported so far (Non-patent Documents 27 and 28).

  In this way, since the CDR sequence of an antibody is deeply involved in the binding to an antigen, it is common to reduce the binding activity by amino acid substitution of the CDR sequence, and the effect on the binding activity to the antigen by amino acid substitution of the CDR sequence Depends on the type of antibody. In Patent Document 1, an example of controlling the isoelectric point by amino acid substitution in CDR is shown, but it may be possible that the binding activity to the antigen may be reduced depending on the type of antibody. Although methods for improving antibody expression by common amino acid substitutions have been reported, regardless of the type of antibody, improving the binding activity of the antibody to the antigen and greatly increasing the binding activity of the antibody to the antigen There has been no report on a method for removing the T-cell epitope without attenuation. Furthermore, there has been no report on the CDR sequence of an antibody that can be substituted for an amino acid without significantly reducing the binding activity of the antibody to the antigen, regardless of the type of antibody.

  Prior art documents of the present invention are shown below.

Monoclonal antibody successes in the clinic, Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nature Biotechnology 23, 1073-1078 (2005) Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 Apr; 59 (3): 389-96. Presta LG. Engineering of therapeutic antibodies to minimize immunogenicity and optimize function.Adv Drug Deliv Rev. 2006 Aug 7; 58 (5-6): 640-56. Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31; 20 (1): 17-29. Review. Fujii I. Antibody affinity maturation by random mutagenesis.Method Mol Biol. 2004; 248: 345-59. Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations.J Pharm Sci. 2004 Jun; 93 (6): 1390-402. Salfeld JG. Isotype selection in antibody engineering.Nat Biotechnol. 2007 Dec; 25 (12): 1369-72. Hinton PR, Johlfs MG, Xiong JM, Hanestad K, Ong KC, Bullock C, Keller S, Tang MT, Tso JY, Vasquez M, Tsurushita N. Engineered human IgG antibodies with longer serum half-lives in primates.J Biol Chem. 2004 Feb 20; 279 (8): 6213-6. Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1; 176 (1): 346-56 Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. 1997 Jul; 15 (7): 637 -40 Almagro JC, Fransson J. Humanization of antibodies.Front Biosci. 2008 Jan 1; 13: 1619-33. Liu H, Gaza-Bulseco G, Faldu D, Chumsae C, Sun J. Heterogeneity of monoclonal antibodies. J Pharm Sci. 2008 Jul; 97 (7): 2426-47 Chen C, Roberts VA, Rittenberg MB.Generation and analysis of random point mutations in an antibody CDR2 sequence: many mutated antibodies lose their ability to bind antigen.J Exp Med. 1992 Sep 1; 176 (3): 855-66. Chen C, Martin TM, Stevens S, Rittenberg MB .Defective secretion of an immunoglobulin caused by mutations in the heavy chain complementarity determining region 2.J Exp Med. 1994 Aug 1; 180 (2): 577-86 Wiens GD, Heldwein KA, Stenzel-Poore MP, Rittenberg MB.Somatic mutation in VH complementarity-determining region 2 and framework region 2: differential effects on antigen binding and Ig secretion.J Immunol. 1997 Aug 1; 159 (3): 1293 -302. Wiens GD, Lekkerkerker A, Veltman I, Rittenberg MB.Mutation of a single conserved residue in VH complementarity-determining region 2 results in a severe Ig secretion defect.J Immunol. 2001 Aug 15; 167 (4): 2179-86. Zwick MB, Komori HK, Stanfield RL, Church S, Wang M, Parren PW, Kunert R, Katinger H, Wilson IA, Burton DR.The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5.J Virol. 2004 Mar; 78 (6): 3155-61. Komissarov AA, Marchbank MT, Calcutt MJ, Quinn TP, Deutscher SL Site-specific mutagenesis of a recombinant anti-single-stranded DNA Fab. Role of heavy chain complementarity-determining region 3 residues in antigen interaction. J Biol Chem. 1997 Oct 24 ; 272 (43): 26864-70. Gerstner RB, Carter P, Lowman HB.Sequence plasticity in the antigen-binding site of a therapeutic anti-HER2 antibody.J Mol Biol. 2002 Aug 30; 321 (5): 851-62. Vajdos FF, Adams CW, Breece TN, Presta LG, de Vos AM, Sidhu SS. Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis.J Mol Biol. 2002 Jul 5; 320 ( 2): 415-28. Pons J, Rajpal A, Kirsch JF. Energetic analysis of an antigen / antibody interface: alanine scanning mutagenesis and double mutant cycles on the HyHEL-10 / lysozyme interaction.Potein Sci. 1999 May; 8 (5): 958-68. Leong SR, DeForge L, Presta L, Gonzalez T, Fan A, Reichert M, Chuntharapai A, Kim KJ, Tumas DB, Lee WP, Gribling P, Snedecor B, Chen H, Hsei V, Schoenhoff M, Hale V, Deveney J , Koumenis I, Shahrokh Z, McKay P, Galan W, Wagner B, Narindray D, Hebert C, Zapata G. Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation. Cytokine. 2001 Nov 7; 16 (3): 106-19. Xiang J, Srivamadan M, Rajala R, Jia Z.tudy of B72.3 combining sites by molecular modeling and site-directed mutagenesis.Protein Eng. 2000 May; 13 (5): 339-44. Rothe A, Hosse RJ, Power BE. Ribosome display for improved biotherapeutic molecules. Expert Opin Biol Ther. 2006 Feb; 6 (2): 177-87. Schmitz U, Versmold A, Kaufmann P, Frank HG. Phage display: a molecular tool for the generation of antibodies--a review.Placenta. 2000 Mar-Apr; 21 Suppl A: S106-12. Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea RA general method for greatly improving the affinity of antibodies by using combinatorial libraries .. Proc Natl Acad Sci US A. 2005 Jun 14; 102 (24): 8466-71. De Groot AS, Knopp PM, Martin W. De-immunization of therapeutic proteins by T-cell epitope modification. Dev Biol (Basel). 2005; 122: 171-94. http://www.algonomics.com/proteinengineering/tripole_applications.php

WO / 2007/114319 US / 2006/0019342

The present invention has been made in view of such circumstances, and its object is to provide a method for modifying the isoelectric point while retaining the binding activity of a polypeptide containing a variable region of an antibody to an antigen, and plasma of the antibody. To provide a method for controlling a half-life, a pharmaceutical composition containing an antibody with controlled plasma half-life as an active ingredient, and a method for producing the antibody and the pharmaceutical composition containing the antibody as an active ingredient .
The present invention also controls the plasma half-life of the antibody by modifying the amino acid residues exposed on the surface of the CDR region of the anti-IL-6 receptor antibody, anti-glypican 3 antibody, and anti-IL-31 receptor antibody. Method, anti-IL-6 receptor antibody, anti-glypican 3 antibody, and anti-IL-31 receptor antibody whose plasma phase is controlled by modification of amino acid residues, method for producing the antibody, and the antibody as an active ingredient The object is to provide a pharmaceutical composition.
Furthermore, the present invention improves the plasma retention while enhancing the antigen neutralization ability by modifying the amino acid sequences of the variable and constant regions of TOCILIZUMAB, which is a humanized anti-IL-6 receptor IgG1 antibody. A pharmaceutical composition comprising a second-generation molecule superior in TOCILIZUMAB, which exhibits a therapeutic effect with low frequency of administration and improves immunogenicity, safety and physical properties, and pharmaceutical compositions thereof It is another object of the present invention to provide a manufacturing method.

  The inventors of the present invention have made extensive studies on a method for modifying the isoelectric point of a polypeptide containing the variable region of the antibody while retaining the binding activity to the antigen of the variable region. As a result, the present inventors modify the isoelectric point of the amino acid residues constituting the complementarity determining region (CDR) of the variable region of the antibody while retaining the binding activity to the antigen of the variable region. A specific position on the CDR amino acid sequence was found. In addition, the plasma half-life of the polypeptide can be controlled by controlling the isoelectric point of the polypeptide containing the variable region of the antibody, and the heteromultimer can be obtained by utilizing the difference in isoelectric point. It has been found that a polypeptide comprising the variable region of an antibody can be efficiently produced. Specifically, among the amino acid residues in the amino acid sequence constituting the variable region of the antibody, the surface of the antibody molecule without affecting the function or structure of the antibody, such as the binding activity to the antigen of the antibody variable region. A position on a specific CDR amino acid sequence that can regulate the charge of was identified. Furthermore, the present inventors have confirmed that the plasma half-life of the polypeptide containing the variable region of the antibody can be controlled by modifying the isoelectric point by adjusting the antibody surface charge. It was confirmed that the antibody whose phase was controlled actually retained the binding activity to the antigen. Furthermore, the present inventors have confirmed that by controlling the plasma half-life of antibodies, the tumor growth inhibitory effect on cancer cells possessed by antibodies that exhibit cytotoxic activity, including antibodies, is increased. Completed the invention. In addition, it was confirmed that heterodimers consisting of antibodies that bind to two or more different antigens can be separated and purified by modifying the isoelectric point by adjusting the charge of CDR.

  Furthermore, the present inventors modified the amino acid sequence of the variable region and constant region of TOCILIZUMAB, which is a first-generation humanized anti-IL-6 receptor IgG1 antibody, to enhance the retention in plasma while enhancing the drug efficacy. To create a second-generation molecule that is superior to TOCILIZUMAB by reducing the frequency of administration and continually exerting therapeutic effects, improving immunogenicity, safety, and physical properties (stability and uniformity). Toward this end, we conducted intensive research. As a result, the present inventors found a plurality of CDR mutations that improve the binding ability (affinity) to the antigen in the variable region of TOCILIZUMAB, and succeeded in greatly improving the affinity by combining them. In addition, the present inventors succeeded in improving plasma retention by introducing a modification that reduces the isoelectric point of the variable region sequence. The present inventors have also succeeded in reducing the number of T-cell epitope peptides predicted in silico in mouse-derived sequences and variable regions remaining in the TOCILIZUMAB framework, thereby reducing the immunogenicity risk. At the same time, we succeeded in improving the stability at high concentrations. Furthermore, the present inventors show that the constant region of TOCILIZUMAB minimizes the appearance of a new T-cell epitope peptide, shows no binding to the Fcγ receptor, is stable under acidic conditions, and is derived from the disulfide of the hinge region. He succeeded in finding a novel constant region sequence with improved heterogeneity, heterogeneity derived from the C-terminus of the H chain, and stability in a high concentration preparation. By combining these modifications of the CDR region amino acid sequence, the variable region amino acid sequence, and the constant region amino acid sequence, we succeeded in creating a second generation molecule superior to TOCILIZUMAB.

More specifically, the following [1] to [44] are provided.
[1] A method for altering the isoelectric point of a polypeptide comprising a variable region of an antibody while retaining the binding activity to the antigen of the variable region, wherein the isoelectric point is applied to the surface of the complementarity determining region (CDR) of the polypeptide. Modifying the charge of at least one amino acid residue that may be exposed;
[2] The method according to [1], wherein the polypeptide comprising the variable region of the antibody further comprises an FcRn binding region,
[3] The method according to [1], wherein the polypeptide containing the variable region of the antibody is an IgG antibody,
[4] The method according to [1], wherein the polypeptide containing the variable region of the antibody is a chimeric antibody, a humanized antibody or a human antibody,
[5] The method according to [1], wherein the polypeptide containing the variable region of the antibody is a multispecific polypeptide that binds to at least two types of antigens,
[6] The method according to [1], wherein the modification of the charge of the amino acid residue is an amino acid substitution,
[7] The method according to [1], wherein the modification of the charge of the amino acid residue is a modification that changes the theoretical isoelectric point by 1.0 or more.
[8] The amino acid residues that can be exposed on the surface of the CDR region are the 31st, 61st, 62nd, 64th and 65th amino acid residues by Kabat numbering in the heavy chain variable region or Kabat numbering in the light chain variable region The method according to [1], which is at least one amino acid residue selected from amino acid residues at positions 24, 27, 53, 54, and 55 according to
[9] A polypeptide comprising a variable region of an antibody with a modified isoelectric point, obtained by the method according to any one of [1] to [8],
[10] A method for controlling plasma pharmacokinetics of the polypeptide, comprising modifying the isoelectric point of the polypeptide containing the variable region of the antibody by the method according to any one of [1] to [8],
[11] The control of the pharmacokinetics is an increase or decrease in any of the parameters of plasma clearance (CL), area under the concentration curve (AUC), mean plasma residence time, and plasma half-life (t1 / 2). The method according to [10],
[12] A polypeptide comprising a variable region of an antibody with controlled plasma pharmacokinetics, obtained by the method according to [10],
[13] A method for producing a polypeptide comprising an antibody variable region with a modified isoelectric point,
(a) modifying a nucleic acid encoding a polypeptide containing the amino acid residue such that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide is modified;
(b) culturing the host cell so that the nucleic acid is expressed;
(c) recovering the polypeptide comprising the antibody variable region from the host cell culture,
[14] The method according to [13], wherein the polypeptide comprising the variable region of the antibody further comprises an FcRn binding region,
[15] The method according to [13], wherein the polypeptide containing the variable region of the antibody is an IgG antibody,
[16] The method according to [13], wherein the polypeptide containing the variable region of the antibody is a chimeric antibody, a humanized antibody or a human antibody,
[17] The method according to [13], wherein the polypeptide containing the variable region of the antibody is a multispecific polypeptide that binds to at least two kinds of antigens,
[18] The method according to [13], wherein the modification of the charge of the amino acid residue is an amino acid substitution,
[19] The method according to [13], wherein the modification of the charge of the amino acid residue is a modification that changes the theoretical isoelectric point by 1.0 or more.
[20] The amino acid residues that can be exposed on the surface of the CDR region are the 31st, 61st, 62nd, 64th and 65th amino acid residues by Kabat numbering in the heavy chain variable region or Kabat numbering in the light chain variable region The method according to [13], which is at least one amino acid residue selected from amino acid residues at positions 24, 27, 53, 54, and 55 according to
[21] A polypeptide comprising a variable region of an antibody with a modified isoelectric point, obtained by the method according to any one of [13] to [20],
[22] A polypeptide containing an antibody variable region with controlled plasma pharmacokinetics, comprising modifying the isoelectric point of a polypeptide containing the antibody variable region by the method according to any one of [13] to [20] A method for producing a peptide,
[23] The control of the pharmacokinetics is an increase or decrease in any of the parameters of plasma clearance (CL), area under the concentration curve (AUC), mean plasma residence time, plasma half-life (t1 / 2). The method according to [22],
[24] A polypeptide comprising a variable region of an antibody with controlled plasma pharmacokinetics produced by the method according to [22],
[25] A method for producing a multispecific polypeptide comprising a first polypeptide having a variable region of an antibody and a second polypeptide,
(A) modifying a nucleic acid encoding a polypeptide containing the amino acid residue such that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide is modified, The nucleic acid encoding the amino acid residue of the first polypeptide and the second polypeptide such that the difference in isoelectric point between the first polypeptide and the second polypeptide is increased compared to that before the modification. Modifying both or either of the nucleic acids encoding the amino acid residues of the polypeptide;
(B) culturing the host cell such that the nucleic acid is expressed;
(C) recovering the multispecific antibody from the host cell culture,
[26] The method according to [25], wherein the step of recovering the multispecific polypeptide containing the first polypeptide and the second polypeptide from the host cell culture is performed by standard chromatography,
[27] In the nucleic acid modification, a standard chromatogram of a first polypeptide homomultimer, a second polypeptide homomultimer, and a heteropolymer of the first polypeptide and the second polypeptide. The method according to [25], wherein the nucleic acid is modified such that the peak obtained by analysis using a graph is a more separated peak than before modification,
[28] The method according to [25], wherein the multispecific polypeptide is a multispecific antibody,
[29] A multispecific antibody produced by the method according to [27],
[30] The multispecific antibody according to [29], wherein the multispecific antibody is a bispecific antibody,
[31] a human-derived CDR, a CDR derived from a non-human animal, and a CDR selected from the group consisting of a synthetic CDR, a human-derived framework region (FR), and an antibody comprising a human constant region, The at least one amino acid residue that can be exposed on the surface of the CDR is an amino acid residue having a different charge from the amino acid residue at the corresponding position of the wild-type CDR, and compared with the antibody before modification, the binding activity to the antigen An antibody with a modified isoelectric point while retaining
[32] The antibody according to [31], wherein the human constant region includes a human Fc region,
[33] The antibody according to [31], wherein the pharmacokinetics in plasma is controlled by altering the isoelectric point,
[34] Amino acid residues at positions 31, 61, 62, 64, and 65 by Kabat numbering in the heavy chain variable region, or positions 24, 27, 53, 54 by Kabat numbering in the light chain variable region An IgG antibody in which the charge of at least one amino acid residue selected from the amino acid residues at position 55 and 55 is modified, and the isoelectric point is modified compared to before the modification of the amino acid residue,
[35] The antibody according to [34], wherein the modified amino acid residue is selected from amino acid residues included in any of the following groups (a) or (b):
(a) glutamic acid (E), aspartic acid (D);
(b) lysine (K), arginine (R), histidine (H),
[36] A multispecific antibody comprising a first polypeptide and a second polypeptide, wherein the positions 31, 61, 62, 64, and 64 by Kabat numbering in the heavy chain variable region of the first polypeptide Amino acid residue at position 65 or at least one amino acid residue selected from amino acid residues at positions 24, 27, 53, 54 and 55 by Kabat numbering in the light chain variable region has a charge A multispecific antibody wherein the isoelectric points of the first polypeptide and the second polypeptide are different from each other;
[37] Amino acid residues at positions 31, 61, 62, 64 and 65 by Kabat numbering in the heavy chain variable region of the second polypeptide, or positions 24 and 27 by Kabat numbering in the light chain variable region The charge of at least one amino acid residue selected from the amino acid residues at positions 53, 54, and 55 has a charge opposite to that of the amino acid residue selected in the first polypeptide Or the antibody according to [36] having no charge,
[38] A combination of an amino acid residue having the above charge and an amino acid residue having a charge opposite to the amino acid residue is selected from the following amino acid residues included in any of the groups (a) or (b): The antibody of [36] selected;
(c) glutamic acid (E), aspartic acid (D);
(d) lysine (K), arginine (R), histidine (H),
[39] A multispecific antibody comprising the first polypeptide and the second polypeptide, wherein a homomultimer of the first polypeptide and a homomultimer of the second polypeptide are subjected to standard chromatography. The antibody according to [36], which results in a peak separated by analysis using
[40] A composition comprising the antibody according to [31] to [39] and a pharmaceutically acceptable carrier,
[41] A nucleic acid encoding the polypeptide constituting the antibody according to [31] to [39],
[42] A host cell having the nucleic acid according to [41],
[43] A method for producing the antibody according to [31] to [39], comprising the step of culturing the host cell according to [42], the step of recovering the polypeptide from the cell culture,
[44] A method for substituting an amino acid residue that can be exposed on the surface of a complementarity determining region (CDR) of a polypeptide in a polypeptide containing a variable region of the antibody while retaining the binding activity of the polypeptide to an antigen At least positions 31, 61, 62, 64 and 65 by Kabat numbering in the heavy chain variable region, or positions 24, 27, 53 by Kabat numbering in the light chain variable region , A method for substituting at least one amino acid residue selected from amino acid residues at positions 54 and 55.

The present invention also provides [1] to [38] below.
[1] A method for producing anti-glypican 3 antibody with controlled blood kinetics comprising the following steps;
(A) modifying a nucleic acid encoding at least one amino acid residue that results in a charge modification of at least one amino acid residue that may be exposed on the surface of the anti-glypican 3 antibody;
(B) culturing a host cell retaining the nucleic acid so that the nucleic acid is expressed;
(C) a method comprising recovering glypican 3 antibody from the host cell culture;
[2] The method according to [1], wherein the control of blood kinetics is extension or reduction of any of the parameters of blood half-life, average blood residence time, blood clearance,
[3] The method according to [1], wherein the charge modification of the amino acid residue in step (a) is performed by amino acid substitution.
[4] The method according to [1], wherein the amino acid residue that can be exposed on the surface of the glypican 3 antibody is in a region other than the FcRn binding region in the glypican 3 antibody,
[5] The method according to [4], wherein the FcRn binding region comprises an Fc region,
[6] The method according to [1], wherein the anti-glypican 3 antibody is an IgG antibody,
[7] The method according to [6], wherein the amino acid residue whose charge is modified is an amino acid residue of a heavy chain variable region or a light chain variable region of an IgG antibody,
[8] The anti-glypican 3 antibody comprising a complementarity determining region (CDR), a human-derived framework region (FR) and a human constant region, wherein the modification of the amino acid residue charge in step (a) Is a modification from at least one amino acid residue that can be exposed on the antibody surface in the CDR or FR of the antibody to be modified to an amino acid residue having a different charge from the amino acid residue [7 The method described in
[9] The method according to [8], wherein the anti-glypican 3 antibody is an antibody having a reduced content of fucose bound to its Fc region,
[10] Glypican 3 antibody produced by the method according to [1] to [9],
[11] At least one of the anti-glypican 3 antibodies that causes an increase in the Tm value of the anti-glypican 3 antibody comprising the complementarity determining region (CDR), human-derived framework region (FR) and human constant region comprising the following steps: A method of stabilizing a glypican 3 antibody characterized by modification of one amino acid residue;
(A) modifying a nucleic acid encoding at least one amino acid residue that causes an increase in the Tm value of the anti-glypican 3 antibody to be modified;
(B) culturing a host cell retaining the nucleic acid so that the nucleic acid is expressed;
(C) a method comprising recovering an antibody from the host cell culture;
[12] The method according to [11], wherein the amino acid residue in step (a) is present in the FR1 region or / and FR2 region of the H chain or L chain thereof,
[13] The method according to [12], wherein the amino acid residue of the FR2 region of the H chain according to [12] is substituted with an amino acid residue of the FR2 region of the VH4 subclass,
[14] The method according to [12], wherein the amino acid residue in the FR2 region of the L chain according to [12] is substituted with an amino acid residue in the FR2 region of the VK3 subclass,
[15] A method for controlling the cytotoxic activity of an antibody comprising the following steps;
(A) modifying a nucleic acid encoding at least one amino acid residue that results in a charge modification of at least one amino acid residue that can be exposed on the surface of an antibody having cytotoxic activity;
(B) culturing a host cell retaining the nucleic acid so that the nucleic acid is expressed;
(C) a method comprising recovering an antibody from the host cell culture;
[16] The method according to [15], wherein the control of blood kinetics is control of any of the parameters of blood half-life, average blood residence time, blood clearance,
[17] The method according to [15], wherein the modification of the charge of the amino acid residue in step (a) is by amino acid substitution,
[18] The method according to [15], wherein the amino acid residue that can be exposed on the surface of the antibody is in a region other than the FcRn binding region in the antibody,
[19] The method according to [18], wherein the FcRn binding region comprises an Fc region,
[20] The method according to [15], wherein the anti-glypican 3 antibody is an IgG antibody,
[21] The method according to [20], wherein the amino acid residue whose charge is modified is an amino acid residue of a heavy chain variable region or a light chain variable region of an IgG antibody,
[22] An antibody comprising a complementarity determining region (CDR) derived from a non-human animal, a framework region (FR) derived from a human and a human constant region, wherein the charge of the amino acid residue in step (a) The modification of is characterized in that at least one amino acid residue that can be exposed on the antibody surface in the CDR or FR of the antibody to be modified is an amino acid residue having a different charge from the amino acid residue. [21] The method according to
[23] The method according to [22], wherein the content of fucose bound to the Fc region of the antibody is reduced.
[24] An antibody produced by the method according to [15] to [23],
[25] The antibody according to [24], wherein the antibody is an anti-glypican 3 antibody,
[26] any one or more of the following substitutions for the amino acid residues constituting the H chain V region represented by SEQ ID NO: 195;
(A) substitution of 19th amino acid residue K with T,
(B) substitution of Q, which is the 43rd amino acid residue, with E,
(C) substitution of S, which is the 63rd amino acid residue, with S;
(D) substitution of the 65th amino acid residue K to Q;
(E) substitution of G, which is the 66th amino acid residue, with D,
H chain V region subjected to, and
Any one or more of the following substitutions for the amino acid residues constituting the L chain V region represented by SEQ ID NO: 201;
(F) Substitution of Q, which is the 27th amino acid residue, to E,
(G) substitution of K, which is the 79th amino acid residue, to T;
(H) substitution of R at the 82nd amino acid residue with S;
L chain V region,
An antibody comprising,
[27] The antibody according to [26], comprising an H chain represented by SEQ ID NO: 197 and an L chain represented by SEQ ID NO: 203,
[28] The antibody according to [26], comprising an H chain represented by SEQ ID NO: 198 and an L chain represented by SEQ ID NO: 204,
[29] any one or more of the following substitutions for the amino acid residues constituting the H chain V region represented by SEQ ID NO: 195;
(A) substitution of Q, which is the 43rd amino acid residue, with K;
(B) substitution of N which is the 52nd amino acid residue with N;
(C) substitution of Q, which is the 107th amino acid residue, with R,
H chain V region subjected to, and
Any one or more of the following substitutions for the amino acid residues constituting the L chain V region represented by SEQ ID NO: 201;
(D) substitution of E as the 17th amino acid residue with Q,
(E) substitution of Q, which is the 27th amino acid residue, with R,
(F) substitution of Q as R at the 105th amino acid residue;
L chain V region,
An antibody comprising,
[30] The antibody according to [29], comprising an H chain variable region represented by SEQ ID NO: 198 and an L chain variable region represented by SEQ ID NO: 204,
[31] The antibody according to [29], comprising an H chain variable region represented by SEQ ID NO: 199 and an L chain variable region represented by SEQ ID NO: 205,
[32] The antibody according to [26] to [31], which has a C region of a human antibody,
[33] A composition comprising the antibody according to [32] and a pharmaceutically acceptable carrier,
[34] A cancer therapeutic agent comprising the antibody according to [32] as an active ingredient,
[35] The cancer therapeutic agent according to [34], wherein the cancer is liver cancer,
[36] A nucleic acid encoding the polypeptide constituting the antibody according to [26] to [31],
[37] a host cell carrying the nucleic acid according to [36],
[38] The method for producing an antibody according to [26] to [31], comprising a step of culturing the host cell according to [37] and a step of recovering the polypeptide from the cell culture.

The present invention also provides [1] to [41] below.
[1] The anti-IL-6 receptor antibody according to any one of the following (a) to (y);
(a) an antibody comprising a heavy chain variable region having CDR1 in which the first Ser is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 1,
(b) an antibody comprising a heavy chain variable region having CDR1 in which the fifth Trp is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 1,
(c) an antibody comprising a heavy chain variable region having CDR2 in which the first Tyr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 2;
(d) an antibody comprising a heavy chain variable region having CDR2 in which the eighth Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 2;
(e) an antibody comprising a heavy chain variable region having CDR2 in which the 9th Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 2;
(f) an antibody comprising a heavy chain variable region having CDR3 in which the first Ser is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 3;
(g) an antibody comprising a heavy chain variable region having CDR3 in which the second Leu is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 3;
(h) an antibody comprising a heavy chain variable region having CDR3 in which the fifth Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 3;
(i) an antibody comprising a heavy chain variable region having CDR3 in which the seventh Ala in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid,
(j) an antibody comprising a heavy chain variable region having CDR3 in which the 8th Met is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 3;
(k) an antibody comprising a heavy chain variable region having CDR3 in which the first Ser and the fifth Thr are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 3;
(l) an antibody comprising a heavy chain variable region having CDR3 in which the second Leu, the seventh Ala and the eighth Met are substituted with other amino acids in the amino acid sequence shown in SEQ ID NO: 3;
(m) an antibody comprising a light chain variable region having CDR1 in which the first Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 4;
(n) an antibody comprising a light chain variable region having CDR1 in which the fourth Gln is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 4;
(o) an antibody comprising a light chain variable region having CDR1 in which the 9th Tyr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 4;
(p) an antibody comprising a light chain variable region having CDR1 in which the 11th Asn in the amino acid sequence of SEQ ID NO: 4 is substituted with another amino acid,
(q) an antibody comprising a light chain variable region having CDR2 in which the second Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 5;
(r) an antibody comprising a light chain variable region having CDR3 in which the first Gln is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 6;
(s) an antibody comprising a light chain variable region having CDR3 in which the third Gly is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 6;
(t) CDR1 in which the 9th Tyr is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 4 and the third Gly in the amino acid sequence described in SEQ ID NO: 6 are replaced with another amino acid An antibody comprising a light chain variable region having CDR3,
(u) an antibody comprising a light chain variable region having CDR3 in which the fifth Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 6;
(v) an antibody comprising a light chain variable region having CDR3 in which the first Gln and the fifth Thr are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 6;
(w) CDR2 in which the 9th Thr is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 2, and the 1st Ser and 5th Thr in the amino acid sequence described in SEQ ID NO: 3 An antibody comprising a heavy chain variable region having CDR3 substituted with an amino acid of
(x) an antibody comprising the heavy chain variable region according to (k) and the light chain variable region according to (v), or
(y) the antibody according to (x), further comprising CDR2 of (e),
[2] an anti-IL-6 receptor antibody comprising a light chain variable region having CDR2 in which the second Thr in the amino acid sequence of SEQ ID NO: 5 is substituted with another amino acid,
[3] The anti-IL-6 receptor antibody according to any one of the following (a) to (y);
(a) an antibody comprising a heavy chain variable region having FR1 in which the 13th Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 7;
(b) an antibody comprising a heavy chain variable region having FR1 in which the 16th Gln is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 7;
(c) an antibody comprising a heavy chain variable region having FR1 in which the 23rd Thr is substituted with another amino acid in the amino acid disruption described in SEQ ID NO: 7;
(d) an antibody comprising a heavy chain variable region having FR1 in which the 30th Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 7;
(e) an antibody comprising a heavy chain variable region having FR1 in which the 13th Arg, 16th Gln, 23rd Thr and 30th Thr are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 7 ,
(f) an antibody comprising a heavy chain variable region having FR2 in which the 8th Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 8;
(g) an antibody comprising a heavy chain variable region having FR3 in which the fourth Met is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 9;
(h) an antibody comprising a heavy chain variable region having FR3 in which the fifth Leu is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 9;
(i) an antibody comprising a heavy chain variable region having FR3 in which the 16th Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 9;
(j) an antibody comprising a heavy chain variable region having FR3 in which the 27th Val is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 9;
(k) an antibody comprising a heavy chain variable region having FR3 in which the 4th Met, 5th Leu, 16th Arg and 27th Val are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 9 ,
(l) an antibody comprising a heavy chain variable region having FR4 in which the third Gln is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 10;
(m) an antibody comprising a light chain variable region having FR1 in which the 18th Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 11;
(n) an antibody comprising a light chain variable region having FR2 in which the 11th Lys is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 12;
(o) an antibody comprising a light chain variable region having FR3 in which the 23rd Gln is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 13;
(p) an antibody comprising a light chain variable region having FR3 in which the 24th Pro is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 13;
(q) an antibody comprising a light chain variable region having FR3 in which the 27th Ile is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 13;
(r) an antibody comprising a light chain variable region having FR3 in which the 23rd Gln, the 24th Pro, and the 27th Ile are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 13;
(s) an antibody comprising a light chain variable region having FR4 in which the 10th Lys is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 14;
(t) an antibody comprising a heavy chain variable region having FR4 in which the fifth Ser in the amino acid sequence of SEQ ID NO: 10 is substituted with another amino acid,
(u) an antibody comprising a heavy chain variable region having FR4 in which the third Gln and the fifth Ser are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 10;
(v) an antibody comprising a heavy chain variable region having FR3 having the amino acid sequence of SEQ ID NO: 184,
(w) FR1 as described in (e), FR2 as described in (f), FR3 as described in (k) and an antibody comprising a heavy chain variable region comprising FR4 as described in (l) or (u),
(x) an antibody comprising a light chain variable region comprising FR1 as described in (m), FR2 as described in (n), FR3 as described in (r) and FR4 as described in (s), or
(y) an antibody comprising the heavy chain variable region according to (w) and the light chain variable region according to (x),
[4] The anti-IL-6 receptor antibody according to any one of the following (a) to (l);
(a) an antibody comprising a heavy chain variable region having CDR1 in which the first Ser is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 1,
(b) an antibody comprising a heavy chain variable region having CDR2 in which the 9th Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 2;
(c) an antibody comprising a heavy chain variable region having CDR2 in which the 16th Ser in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid,
(d) an antibody comprising a heavy chain variable region having CDR2 in which the 9th Thr and the 16th Ser are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 2;
(e) an antibody comprising a light chain variable region having CDR1 in which the first Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 4;
(f) an antibody comprising a light chain variable region having CDR2 in which the second Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 5;
(g) an antibody comprising a light chain variable region having CDR2 in which the fourth Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 5;
(h) an antibody comprising a light chain variable region having CDR2 in which the second Thr and the fourth Arg are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 5;
(i) an antibody comprising a light chain variable region having CDR3 in which the fifth Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 6;
(j) an antibody comprising a heavy chain variable region comprising CDR1 of (a), CDR2 of (d) and CDR3 having the amino acid sequence of SEQ ID NO: 3,
(k) an antibody comprising a light chain variable region comprising CDR1 according to (e), CDR2 according to (h) and CDR3 according to (i), or
(l) an antibody comprising the heavy chain variable region according to (j) and the light chain variable region according to (k),
[5] The anti-IL-6 receptor antibody according to any one of the following (a) to (f):
(a) CDR1 in which the first Ser is substituted with another amino acid sequence in the amino acid sequence described in SEQ ID NO: 1, and the 9th Thr and 16th Ser in the amino acid sequence described in SEQ ID NO: 2 are the other An antibody comprising CDR2 substituted with an amino acid, and a heavy chain variable region comprising CDR3 in which the first Ser and the fifth Thr are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 3;
(b) CDR1 in which the first Arg is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 4, and the second Thr and the fourth Arg in the amino acid sequence described in SEQ ID NO: 5 are other amino acids. An antibody comprising a light chain variable region comprising CDR2 substituted with, and CDR3 in which the first Gln and the fifth Thr are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 6;
(c) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 22;
(d) an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 23,
(e) an antibody comprising the heavy chain variable region according to (a) and the light chain variable region according to (b),
Or
(f) an antibody comprising the heavy chain variable region according to (c) and the light chain variable region according to (d),
[6] The human antibody constant region described in any of (a) to (c) below;
(a) A human antibody constant characterized by deletion of Gly at position 329 (EU numbering 446) and Lys at position 330 (EU numbering 447) in the amino acid sequence set forth in SEQ ID NO: 19 region,
(b) A human antibody constant characterized by deletion of both 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys in the amino acid sequence of SEQ ID NO: 20. region,
(c) A human antibody constant characterized by deletion of both 326th (EU numbering 446th) Gly and 327th (EU numbering 447th) Lys in the amino acid sequence of SEQ ID NO: 21 region,
[7] In the amino acid sequence set forth in SEQ ID NO: 20, the amino acid at position 209 (EU numbering 330th), position 210 (EU numbering 331) and position 218 (EU numbering 339) was substituted with another amino acid. IgG2 constant region,
[8] IgG2 constant region in which the amino acid at position 276 (EU numbering 397) in the amino acid sequence set forth in SEQ ID NO: 20 is substituted with another amino acid;
[9] In the amino acid sequence set forth in SEQ ID NO: 20, the 14th (EU numbering 131st), 102th (EU numbering 219th), and / or 16th (EU numbering 133th) amino acid is the other amino acid. A substituted IgG2 constant region,
[10] The amino acid sequence of SEQ ID NO: 20 is characterized in that the 20th (EU numbering 137th) and 21st (EU numbering 138th) amino acids are further substituted with other amino acids, [ 9], the IgG2 constant region,
[11] 147th (EU numbering 268th) His, 234th (EU numbering 355th) Arg and / or 298th (EU numbering 419th) in the amino acid sequence of SEQ ID NO: 20 IgG2 constant region in which Gln is replaced with another amino acid,
[12] In the amino acid sequence set forth in SEQ ID NO: 20, the 209th (EU numbering 330th), 210th (EU numbering 331th), 218th (EU numbering 339th), 276th (EU numbering 397th), The 14th (EU numbering 131), 16th (EU numbering 133th), 102th (EU numbering 219th), 20th (EU numbering 137th) and 21st (EU numbering 138th) amino acids are other amino acids. IgG2 constant region having the amino acid sequence substituted with
[13] IgG2 constant region according to [12], further having an amino acid sequence in which 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys are deleted,
[14] In the amino acid sequence described in SEQ ID NO: 20, the 276th (EU numbering 397th), the 14th (EU numbering 131st), the 16th (EU numbering 133th), the 102nd (EU numbering 219th), IgG2 constant region having an amino acid sequence in which the 20th (EU numbering 137th) and 21st (EU numbering 138th) amino acids are substituted with other amino acids,
[15] In the IgG2 constant region of [14], an IgG2 constant region further comprising an amino acid sequence in which 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys are deleted,
[16] In the amino acid sequence of SEQ ID NO: 20, the 14th (EU numbering 131st) Cys, the 16th (EU numbering 133rd) Arg, the 102nd (EU numbering 219th) Cys, the 20th (EU numbering 137th) Glu, 21st (EU numbering 138th) Ser, 147th (EU numbering 268th) His, 234th (EU numbering 355th) Arg and 298th (EU numbering 419th) IgG2 constant region having an amino acid sequence in which Gln is substituted with another amino acid,
[17] In the IgG2 constant region of [16], an IgG2 constant region having an amino acid sequence further lacking 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys,
[18] In the amino acid sequence shown in SEQ ID NO: 20, the 14th (EU numbering 131st) Cys, the 16th (EU numbering 133th) Arg, the 102nd (EU numbering 219th) Cys, the 20th (EU numbering 137th) Glu, 21st (EU numbering 138th) Ser, 147th (EU numbering 268th) His, 234th (EU numbering 355th) Arg, 298th (EU numbering 419th) Gln, and IgG2 constant region having an amino acid sequence in which 313rd (EU numbering 434th) Asn is substituted with another amino acid,
[19] In the IgG2 constant region of [18], an IgG2 constant region having an amino acid sequence in which 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys are deleted,
[20] An IgG4 constant region, wherein the amino acid at position 289 (EU numbering 409) is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 21;
[21] In the amino acid sequence set forth in SEQ ID NO: 21, 289th (EU numbering 409th), 14th, 16th, 20th, 21st, 97th, 100th, 102nd, 103rd, 104th and An amino acid sequence in which the amino acid at position 105 (EU numbering 131,133,137,138,214,217,219,220,221,222), 113th, 114th and 115th (EU numbering 233,234,235) is substituted with another amino acid and the amino acid at position 116 (EU numbering 236) is deleted. IgG4 constant region having,
[22] The IgG4 constant region described in [21], wherein the 326th (EU numbering 446th) Gly and the 327th (EU numbering 447th) Lys are deleted,
[23] In the amino acid sequence set forth in SEQ ID NO: 20, 209th (EU numbering 330th) Ala, 210th (EU numbering 331st) Pro, 218th (EU numbering 339th) Thr, 14th (131th EU numbering) Cys, 16th (133th EU numbering) Arg, 102nd (219th EU numbering) Cys, 20th (137th EU numbering) Glu, 21st IgG2 constant region having an amino acid sequence in which Ser (EU numbering No. 138) is replaced with another amino acid,
[24] The IgG2 constant region according to [23], further having an amino acid sequence in which 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys are deleted,
[25] Cys at position 14 (EU numbering 131), Arg at position 16 (EU numbering 133), Cys at position 102 (EU numbering 219), 20th position (EU numbering 137) ) Glu, IgG2 constant region having an amino acid sequence in which the 21st (EU numbering 138) Ser is replaced with another amino acid,
[26] IgG2 constant region according to [25], further having an amino acid sequence in which 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys are deleted,
[27] A constant region having the amino acid sequence of SEQ ID NO: 24,
[28] A constant region having the amino acid sequence of SEQ ID NO: 118,
[29] A constant region having the amino acid sequence of SEQ ID NO: 25,
[30] A constant region having the amino acid sequence of SEQ ID NO: 151,
[31] A constant region having the amino acid sequence of SEQ ID NO: 152,
[32] A constant region having the amino acid sequence of SEQ ID NO: 153,
[33] a constant region having the amino acid sequence of SEQ ID NO: 164,
[34] a human antibody constant region having the amino acid sequence set forth in SEQ ID NO: 194 (M40ΔGK),
[35] a human antibody constant region having the amino acid sequence of SEQ ID NO: 192 (M86ΔGK),
[36] An antibody having the constant region according to any one of [6] to [35],
[37] The antibody according to [36], which binds to an IL-6 receptor,
[38] an anti-human IL-6 receptor antibody having an IL-6 receptor binding activity of 1 nM or less,
[39] An anti-human IL-6 receptor antibody having a measured isoelectric point of 7.0 or less for a full-length antibody or a theoretical isoelectric point of a variable region of 5.0 or less,
[40] The increase in the antibody aggregate ratio after 1 month at 25 ° C. under the condition that the antibody concentration is 100 mg / mL in a buffer solution of 20 mM Histidine-HCl, 150 mM NaCl, pH 6.5 to 7.0 % Anti-IL-6 receptor antibody,
[41] A pharmaceutical composition comprising the antibody according to any one of [36] to [40].

It is a graph which shows the BaF / gp130 neutralization activity of WT and RD_6. It is a graph which shows the sensorgram of the interaction of rhIL-s6R (R & D systems) and WT. It is a graph which shows the sensorgram of the interaction of rhIL-s6R (R & D systems) and RD_6. It is a figure which shows the list | wrist of the CDR variation | mutation which affinity / neutralization activity improves compared with WT. It is a figure which shows the continuation of FIG. It is a figure which shows the list | wrist of the CDR variation | mutation which affinity / neutralization activity improves by combination. It is a graph which shows the BaF / gp130 neutralization activity of WT and RDC23. It is a graph which shows the sensorgram of the interaction of rhIL-s6R (R & D systems) and RDC23. It is a graph which shows the sensorgram of the interaction of rhsIL-6R and WT. It is a graph which shows the sensorgram of the interaction of rhsIL-6R and RDC23. It is a graph which shows the sensorgram of interaction of SR344 and WT. It is a graph which shows the sensorgram of interaction of SR344 and RDC23. It is a graph which shows the BaF / gp130 neutralization activity of WT and H53L28. It is a graph which shows the sensorgram of interaction of SR344 and H53 / L28. 2 is a graph showing changes in plasma concentration after intravenous administration of WT and H53 / L28 to mice. It is the graph which showed the plasma concentration transition after subcutaneous administration of WT and H53 / L28 to a mouse. It is a graph which shows the BaF / gp130 neutralization activity of WT and PF1. It is a graph which shows the sensorgram of interaction of SR344 and PF1. It is a graph which shows the high concentration stability test result of WT and PF1. 2 is a graph showing changes in plasma concentrations after intravenous administration of WT and PF1 to human IL-6 receptor transgenic mice. 3 is a graph showing changes in the concentration of unbound human soluble IL-6 receptor after intravenous administration of WT and PF1 to human IL-6 receptor transgenic mice. 3 is a graph showing the analysis results of aggregate content by gel filtration chromatography of WT-IgG1, WT-IgG2, WT-IgG4, IgG2-M397V, and IgG4-R409K purified by the hydrochloric acid elution method. It is a figure which shows the cation exchange chromatography (IEC) analysis result of WT-IgG1, WT-IgG2, and WT-IgG4. It is a figure which shows the putative disulfide bond mode of the hinge region of WT-IgG2. It is a figure which shows the putative disulfide bond mode of the hinge region of WT-IgG2-SKSC. It is a figure which shows the cation exchange chromatography (IEC) analysis result of WT-IgG2 and IgG2-SKSC. It is a figure which shows the cation exchange chromatography (IEC) analysis result of humanized PM1 antibody, H chain C terminal (DELTA) K antibody, and H chain C terminal (DELTA) GK antibody. It is a figure which shows the comparison with the binding amount with respect to FcγRI of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, WT-M11ΔGK. It is a graph which shows the comparison of the binding amount with respect to FcγRIIa of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK and WT-M11ΔGK. It is a graph which shows the comparison of the binding amount with respect to FcγRIIb of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK and WT-M11ΔGK. It is a graph which shows the comparison of the binding amount with respect to FcγRIIIa (Val) of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, WT-M11ΔGK. It is a graph which shows the aggregate increase amount in the high concentration stability test of WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK. It is a graph which shows the Fab fragment increase amount in the high concentration stability test of WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK. It is a figure which shows the cation exchange chromatography (IEC) analysis result of WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK. It is a graph which shows the BaF / gp130 neutralization activity of WT and F2H / L39-IgG1. It is a graph which shows antibody plasma concentration transition when WT, PF1, and F2H / L39-IgG1 are subcutaneously administered at 1.0 mg / kg in cynomolgus monkeys. It is a graph which shows CRP density | concentration transition of the WT and F2H / L39-IgG1 administration group in a cynomolgus monkey. It is a graph which shows a non-binding type | mold cynomolgus IL-6 receptor concentration transition of the WT and F2H / L39-IgG1 administration group in a cynomolgus monkey. It is the graph which showed the plasma concentration transition after intravenous administration of WT-IgG1 and WT-M14 to a human FcRn transgenic mouse. It is the graph which showed the plasma concentration transition after intravenous administration of WT-IgG1, WT-M14 and WT-M58 to human FcRn transgenic mice. It is the graph which showed the plasma concentration transition after intravenous administration of WT-IgG1, WT-M44, WT-M58, and WT-M73 to human FcRn transgenic mice. Effect of anti-IL-6 receptor antibody WT, anti-IL-6 receptor antibody F2H / L39, anti-IL-31 receptor antibody H0L0, anti-RANKL antibody DNS constant region on heterogeneity by cation exchange chromatography It is the figure which evaluated. It is the figure which evaluated the influence on the heterogeneity which the cysteine of the CH1 domain of the anti- IL-6 receptor antibody WT and the anti-IL-6 receptor antibody F2H / L39 exerts on the cation exchange chromatography. It is the figure which evaluated the influence on the denaturation peak which the cysteine of the CH1 domain of the anti- IL-6 receptor antibody WT has on DSC. It is a graph which shows neutralizing activity in BaF / gp130 of TOCILIZUMAB, control, and Fv5-M83. It is a graph which shows the neutralizing activity in BaF / gp130 of TOCILIZUMAB, Fv3-M73, and Fv4-M73. FIG. 5 is a graph showing changes in plasma concentration after intravenous administration of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys. It is the graph which showed the CRP density | concentration transition after intravenous administration to TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 to a cynomolgus monkey. It is the graph which showed transition of the neutralization rate of soluble IL-6 receptor after intravenous administration to TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys. It is the chart obtained from DSC (Differential Scanning Calorimetry) measurement of Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody. It is an electrophoretic image of H0L0 antibody and Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody in high pI isoelectric focusing. It is an electrophoretic image of H0L0 antibody and Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody in low pI isoelectric focusing. It is a figure which shows the binding activity with respect to the glypican 3 which is an antigen by competitive ELISA using H15L4 antibody and H0L0 antibody. It is a figure which shows the binding activity with respect to the glypican 3 which is an antigen by competitive ELISA using a Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody and a H0L0 antibody. It is a figure which shows the binding activity with respect to the glypican 3 which is an antigen by competitive ELISA using the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody and the H0L0 antibody. The antitumor effect in the human liver cancer transplant mouse model of H0L0 antibody, Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody, and Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody is shown. 1 shows antibody plasma concentrations of H0L0 antibody, Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody and Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody in a mouse model transplanted with human liver cancer. The ADCC activity by each test antibody with respect to human hepatoma cell line Hep G2 cell is shown. FIG. 6 is a graph showing IL-6 receptor neutralizing activity of BaR / 6R of 6R_b_H1L1, 6R_b_H2L2, 6R_b_H2L3, and 6R_b_H2L4. It is a figure which shows the binding activity with respect to the glypican 3 which is an antigen by competitive ELISA using GPC_H1L1 antibody and GPC_H2L2 antibody. It is a figure which shows the binding activity with respect to the glypican 3 which is an antigen by competitive ELISA using GPC_H2L2 antibody and GPC_H3L3 antibody. It is a figure which shows the peak separation of A chain B chain heterodimer, A chain homodimer, and B chain homodimer by the cation exchange chromatography of 6R_a_H1H3L3, GPC3_H2H3L3, and 31R_H1aH2aL2.

[Embodiment of the Invention]
The present invention relates to a method for modifying the isoelectric point of a polypeptide containing a variable region of an antibody while retaining the binding activity to the antigen of the variable region, the surface of the complementarity determining region (CDR) of the polypeptide A method comprising converting the charge of at least one amino acid residue that may be exposed to The present invention also provides a polypeptide comprising an antibody variable region with a modified isoelectric point obtained by the method (for example, selected from the group consisting of human-derived CDRs, non-human animal CDRs, and synthetic CDRs). An antibody comprising a CDR, a human-derived framework region (FR), and a human constant region, wherein at least one amino acid residue that can be exposed on the surface of the CDR is an amino acid residue at a corresponding position of the wild-type CDR Is an amino acid residue having a different charge, and provides an antibody whose isoelectric point is modified while retaining the binding activity to the antigen as compared to the antibody before modification.

  A preferred embodiment of the method of the present invention is a method comprising modifying the charge of at least one amino acid residue that can be exposed on the surface of an antibody. That is, by altering the charge of an amino acid residue of an antibody and changing its isoelectric point (pI), the plasma pharmacokinetics (blood kinetics) of the antibody can be controlled. As a result, antibodies with controlled plasma pharmacokinetics can exhibit superior antitumor activity against, for example, cancer cells, compared to uncontrolled antibodies.

  In the method of the present invention, maintaining the binding activity to the antigen means having at least 80% or more, preferably 85% or more, particularly preferably 90% or more of the activity compared to the binding activity of the peptide before modification. To do. In addition, when an antibody binds to an antigen, it is sufficient that the binding activity is maintained to such an extent that the function of the antibody is maintained.For example, the affinity measured at 37 ° C. under physiological conditions is 100 nM or less. Preferably, it is 50 nM or less, more preferably 10 nM or less, still more preferably 1 nM or less. Whether or not a polypeptide containing a variable region of an antibody with a modified isoelectric point obtained by the method of the present invention retains an antigen-binding activity can be determined by a known method such as BIACORE (intermolecular interaction analysis). ), Cell proliferation assay, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent immunoassay.

Examples of the “polypeptide containing the variable region of an antibody” of the present invention include, but are not limited to, an antibody, a low molecular weight antibody, a Scaffold protein, and the like.
In the present invention, the Scaffold protein can be used as long as it is a conformationally stable peptide capable of binding to at least one antigen. Examples of such peptides include antibody variable region fragments, fibronectin, protein A domain, LDL receptor A domain, lipocalin and the like, as well as Nygren et al. (Current Opinion in Structural Biology, 7: 463-469 (1997), Journal). of Immunol Methods, 290: 3-28 (2004)), Binz et al. (Nature Biotech 23: 1257-1266 (2005)), Hosse et al. (Protein Science 15: 14-27 (2006)). .

  In the present invention, the term “antibody” is used in the broadest sense. As long as the desired biological activity is exhibited, monoclonal antibodies, polyclonal antibodies, antibody variants (chimeric antibodies, humanized antibodies, low molecular weight antibodies (antibodies) Fragments), multispecific antibodies, and the like. In the present invention, the antibody modification method of the present invention can be preferably used when obtaining (creating) these antibodies.

  In the “antibody” of the present invention, an antibody in which the amino acid sequence is further modified by substitution, deletion, addition, and / or insertion of an amino acid in addition to the antibody in which the charge of the amino acid residue is modified as described above. included. Also included are antibodies in which the amino acid residue has been further modified with respect to antibodies whose amino acid sequence has been modified by amino acid substitution, deletion, addition and / or insertion, chimerization or humanization, etc. . That is, it may be modified simultaneously with the step of humanizing the mouse antibody, or the humanized antibody may be further modified.

  Amino acid substitutions, deletions, additions and / or insertions, and amino acid sequence modifications such as humanization and chimerization can be performed by methods known to those skilled in the art. Similarly, the variable region and constant region of an antibody used when the antibody of the present invention is produced as a recombinant antibody is also replaced by amino acid substitution, deletion, addition and / or insertion, chimerization, humanization, etc. The sequence may be altered.

  The antibody in the present invention may be an antibody derived from any animal such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody. Furthermore, for example, a modified antibody having a substituted amino acid sequence such as a chimeric antibody, particularly a humanized antibody may be used. In addition, any antibody such as a modified antibody obtained by binding various molecules, an antibody fragment, and a low molecular weight antibody may be used.

  A “chimeric antibody” is an antibody produced by combining sequences derived from different animals. For example, an antibody comprising a heavy chain and light chain variable (V) region of a mouse antibody and a human antibody heavy chain and light chain constant (C) region can be exemplified. The production of a chimeric antibody is known. For example, a chimeric antibody is obtained by ligating DNA encoding an antibody V region with DNA encoding a human antibody C region, incorporating it into an expression vector, introducing it into a host, and producing it. be able to.

  Further, the structure, production method and the like of the low molecular weight antibody in the present invention are not particularly limited as long as it has an ability to bind to an antigen. Among the low molecular weight antibodies, there is an antibody having higher activity than the full-length antibody (Orita et al., Blood (2005) 105: 562-566). In the present specification, the “low molecular weight antibody” is not particularly limited as long as it is a part of a full-length antibody (whole antibody such as whole IgG), but is not limited to heavy chain variable region (VH) or light chain variable region (VL). It is preferable that it contains. Examples of preferable antibody fragments include, for example, Fab, F (ab ′) 2, Fab ′, Fv and the like. The amino acid sequence of VH or VL in the antibody fragment may be modified by substitution, deletion, addition and / or insertion. Furthermore, a part of VH and VL may be deleted as long as the antigen-binding ability is maintained. For example, among the above antibody fragments, “Fv” is the smallest antibody fragment containing a complete antigen recognition site and a binding site. “Fv” is a dimer (VH-VL dimer) in which one VH and one VL are strongly bound by a non-covalent bond. An antigen-binding site is formed on the surface of the VH-VL dimer by three complementarity determining regions (CDRs) of each variable region. Six CDRs confer antigen binding sites on the antibody. However, even one variable region (or half of an Fv containing only three CDRs specific for the antigen) has a lower affinity than the entire binding site, but has the ability to recognize and bind to the antigen. . Therefore, such a molecule smaller than Fv is also included in the low molecular weight antibody in the present invention. The variable region of the low molecular weight antibody may be chimerized or humanized.

  The low molecular weight antibody preferably contains both VH and VL. Examples of low molecular weight antibodies include antibody fragments such as Fab, Fab ′, F (ab ′) 2 and Fv, and scFv (single chain Fv) that can be produced using antibody fragments (Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-83; Pluckthun “The Pharmacology of Monoclonal Antibodies” Vol. 113, Resenburg and Moore, Springer Verlag, New York, pp.269-315, (1994) ), Diabody (Holliger et al., Proc. Natl. Acad. Sci. USA (1993) 90: 6444-8; EP404097; WO93 / 11161; Johnson et al., Method in Enzymology (1991) 203: 88- 98; Holliger et al., Protein Engineering (1996) 9: 299-305; Perisic et al., Structure (1994) 2: 1217-26; John et al., Protein Engineering (1999) 12 (7): 597- 604; Atwell et al., Mol. Immunol. (1996) 33: 1301-12), sc (Fv) 2 (Hudson et al, J Immunol. Methods (1999) 231: 177-89; Orita et al., Blood (2005) 105: 562-566), Triabody (Journal of Immunological Methods (1999) 231: 177-89), Tandem Diabody (Cancer Research (2000) 60: 4336-41), etc. It can gel.

  Antibody fragments can be obtained by treating an antibody with an enzyme, for example, a protease such as papain or pepsin (Morimoto et al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al., Science (1985) 229: 81). It can also be produced by gene recombination based on the amino acid sequence of the antibody fragment.

  A low molecular weight antibody having a structure obtained by modifying an antibody fragment can be constructed using an antibody fragment obtained by enzyme treatment or gene recombination. Alternatively, a gene encoding the entire low molecular weight antibody can be constructed, introduced into an expression vector, and then expressed in an appropriate host cell (for example, Co et al., J. Immunol. (1994) 152). : 2968-76; Better and Horwitz, Methods Enzymol. (1989) 178: 476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178: 497-515; Lamoyi, Methods Enzymol. (1986) 121: 652-63 Rousseaux et al., Methods Enzymol. (1986) 121: 663-9; Bird and Walker, Trends Biotechnol. (1991) 9: 132-7).

  The “scFv” is a single-chain polypeptide in which two variable regions are linked via a linker or the like as necessary. The two variable regions included in scFv are usually one VH and one VL, but may be two VHs or two VLs. In general, scFv polypeptides include a linker between the VH and VL domains, thereby forming the VH and VL paired portions necessary for antigen binding. In general, in order to form a paired part between VH and VL in the same molecule, generally, a linker that connects VH and VL is a peptide linker having a length of 10 amino acids or more. However, the scFv linker in the present invention is not limited to such a peptide linker as long as it does not prevent the formation of scFv. As a review of scFv, Pluckthun “The Pharmacology of Monoclonal Antibody” Vol. 113 (Rosenburg and Moore ed., Springer Verlag, NY, pp. 269-315 (1994)) can be referred to.

  In addition, “diabody (Db)” refers to a bivalent antibody fragment constructed by gene fusion (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444- 6448 (1993), EP404,097, WO93 / 11161, etc.). A diabody is a dimer composed of two polypeptide chains, each of which is in a position where the light chain variable region (VL) and heavy chain variable region (VH) cannot bind to each other in the same chain. They are linked by a short linker, for example, about 5 residues. Since VL and VH encoded on the same polypeptide chain cannot form a single-chain V region fragment due to the short linker between them, a diabody forms two antigen-binding sites. Will have. At this time, if VL and VH for two different epitopes (a and b) are combined with VLa-VHb and VLb-VHa and linked together by a linker of about 5 residues, they are secreted as bispecific Db. .

  Diabody contains two molecules of scFv, so it contains four variable regions, resulting in two antigen-binding sites. Unlike the case of scFv that does not form a dimer, when the purpose is to form a diabody, the linker that connects VH and VL in each scFv molecule is usually around 5 amino acids when it is a peptide linker. . However, the scFv linker that forms Diabody is not limited to such a peptide linker as long as it does not prevent scFv expression and does not prevent Diabody formation.

  Of the isotypes of antibodies, IgG has a sufficiently high molecular weight, so its main metabolic pathway is not due to renal excretion. IgG antibodies that have an Fc region as part of their molecules may have a long in vivo half-life by being recycled through the salvage pathway of fetal Fc receptor (FcRn) expressed in endothelial cells such as blood vessels Are known. IgG antibodies are thought to be metabolized mainly by metabolic pathways in endothelial cells (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin. J Immunol. (1998), 160 (2), 1029-35). That is, it is considered that IgG antibodies that are not specifically bound to endothelial cells bind to FcRn, whereby IgG antibodies are recycled, while IgG antibodies that cannot be bound are metabolized. The plasma half-life of an IgG antibody whose Fc portion has been modified so that the binding activity to FcRn is reduced is shortened. Conversely, by modifying amino acid residues constituting the Fc region of IgG antibody in order to increase the binding activity to FcRn, the plasma half-life of IgG antibody can be extended (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL.Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin.J Immunol. (1998), 160 (2), 1029-35). As described above, conventional methods for controlling plasma pharmacokinetics of IgG antibodies have been performed by modifying the binding activity to FcRn by modifying amino acid residues constituting the Fc region. However, as shown in the Examples below, in the present invention, it was revealed that the plasma half-life of the antibody depends on pI with high correlation. That is, it was shown that the plasma half-life of an antibody can be controlled without altering the amino acid sequence that constitutes Fc, which may lead to acquisition of immunogenicity.

  Although not intended to be bound by any particular theory, the present inventors currently consider as follows. The rate of nonspecific IgG antibody uptake into endothelial cells is thought to depend on the physicochemical Coulomb interaction between the negatively charged cell surface and the IgG antibody. Therefore, reducing (increasing) the coulomb interaction by lowering (increasing) the pI of IgG antibodies reduces (increases) non-specific uptake into endothelial cells, resulting in decreased (increased) metabolism in endothelial cells. It is considered that the pharmacokinetics in plasma could be controlled. Since the Coulomb interaction between the endothelial cells and the negative charge on the cell surface is a physicochemical interaction, it is considered that this interaction does not uniquely depend on the amino acid sequence constituting the antibody itself. Therefore, the method for controlling plasma pharmacokinetics found in the present invention is not only applied to specific antibodies, but can be widely applied to any polypeptide containing a variable region of an antibody. Such a peptide is preferably a peptide having a molecular weight of 50,000 or more, more preferably a peptide having a molecular weight of 100,000 or more, and further preferably a peptide having a molecular weight of 140,000 or more. With such a peptide, the main metabolic pathway is not renal excretion, and the plasma pharmacokinetic control effect of the present invention can be sufficiently obtained. Note that the reduction (increase) of the Coulomb interaction in the present invention means an increase (decrease) in the Coulomb force expressed by repulsion.

  The polypeptide containing the FcRn binding region in the present invention is not limited to an IgG antibody, and may be any protein that can bind to an Fc receptor (FcRn) (has binding activity or affinity). Preferably, the polypeptide containing the FcRn binding region in the present invention is a protein containing the Fc region or Fc-like region of an antibody, although not particularly limited. As the Fc region, a modified Fc region can be used. For example, the modified Fc region of J Immunol. (1998), 160 (2), 1029-35 described above can be used. Examples of the polypeptide containing the FcRn binding region in the present invention include IgG antibodies. Even if these antibodies (proteins) are modified, any protein capable of binding to FcRn is included in the polypeptide containing the FcRn-binding region of the present invention. In the present invention, the most preferred example of the polypeptide containing the FcRn binding region is an IgG antibody.

  When an IgG antibody is used as the antibody of the present invention, it may be any subtype as long as it is an IgG type antibody molecule, and may be a multispecific (eg, bispecific) IgG antibody. This bispecific antibody is an antibody having specificity for two different epitopes, and includes antibodies that recognize different antigens as well as antibodies that recognize different epitopes on the same antigen. Even in the case of antibody molecules, in the case of low molecular weight antibodies such as scFv and Fab where renal excretion is the main metabolic pathway, pI cannot control plasma pharmacokinetics as described above. The present invention can be applied to any antibody molecular form as long as it contains a variable region of an antibody whose renal excretion is not a major metabolic pathway, such as scFv-Fc, dAb-Fc, and Fc fusion protein. It is done. Since renal excretion of these molecules is not the main metabolic pathway, it is possible to control plasma pharmacokinetics by changing the pI by the method found in the present invention. The antibody molecule to which the present invention can be applied may be an antibody-like molecule. An antibody-like molecule is a molecule that functions by binding to a target molecule (Binz HK, Amstutz P, Pluckthun A., Engineering novel binding proteins from nonimmunoglobulin domains., Nat Biotechnol. 2005 Oct; 23 ( 10): 1257-68.), For example, DARPins, Affibody, Avimer and the like.

  When the antibody of the present invention is, for example, a bispecific anti-glypican 3 antibody, the antibody can also specifically bind to epitopes of antigens other than glypican 3 and glypican 3. For example, as antigens other than glypican 3, surface antigens that specifically bind to these cells can be suitably used to recruit NK cells, cytotoxic T cells, LAK cells, and the like. Cytotoxic activity of LAK cells was demonstrated against cholangiocarcinoma using bispecific antibody made from MUSE11, an antibody that recognizes MUC1, an adenocarcinoma-related antigen, and OKT3, an antibody that recognizes LAK cell surface antigen (Katayose Y, Kudo T, Suzuki M, Shinoda M, Saijyo S, Sakurai N, Saeki H, Fukuhara K, Imai K, Matsuno S. MUC1-specific targeting immunotherapy with bispecific antibodies: inhibition of xenografted human bile duct carcinoma growth. Cancer Res. (1996) 56 (18), 4205-12). In place of the antibody MUSE11 that recognizes MUC1, the anti-glypican 3 antibody with improved plasma pharmacokinetics provided by the present invention can be preferably used. In addition, as the bispecific anti-glypican 3 antibody provided by the present invention, an antibody that recognizes different epitopes of anti-glypican 3 molecules can be suitably used.

  The “bispecific antibody” may be, for example, an antibody having a structure in which a heavy chain variable region and a light chain variable region are linked as a single chain (for example, sc (Fv) 2). Also, an antibody-like molecule (for example, scFv) in which scFv (or sc (Fv) 2) linked to heavy chain variable region (VH) and light chain variable region (VL) is bound to Fc region (constant region lacking CH1 domain). -Fc). A multispecific antibody consisting of scFv-Fc has a (scFv) 2-Fc type structure in which the first polypeptide is VH1-linker-VL1-Fc and the second polypeptide is VH2-linker-VL2-Fc. It has. Alternatively, it may be an antibody-like molecule in which a single domain antibody is bound to an Fc region (Curr Opin Drug Discov Devel. 2006, 9 (2), 184-93).

In the present invention, the charge of an amino acid residue can be modified through amino acid substitution. Amino acid substitution can be performed by the method described below.
In the present invention, the amino acid residues that can be exposed on the surface of the CDR region to be substituted are positions 31, 61, 62 by Kabat numbering in the heavy chain variable region, from the viewpoint of maintaining the binding activity to the antigen. It is at least one amino acid residue selected from amino acid residues at positions 64, 65, 54, and 55 by amino acid residues at positions 64 and 65 or Kabat numbering in the light chain variable region preferable. The substitution of these amino acid residues retains the functions (such as antigen-binding activity) possessed by the polypeptide containing the variable region of the antibody before the amino acid residue substitution. This is useful in that it does not depend on the type of antibody.

  The present invention also provides a method for controlling the pharmacokinetics of a polypeptide by modifying the isoelectric point of the polypeptide containing the variable region of the antibody. Furthermore, a polypeptide containing a variable region of an antibody with controlled pharmacokinetics obtained by the method is also included in the present invention.

  In the present invention, “plasma pharmacokinetics are controlled” means that the plasma pharmacokinetics of an antibody before and after modification of amino acids constituting the antibody are compared, and the plasma pharmacokinetics are modified in a desired direction. Means that That is, when it is desired to extend the drug (plasma) half-life of an antibody, “control of plasma pharmacokinetics” means that the plasma half-life of an antibody is extended. When it is desired to shorten the plasma half-life of the antibody, “control of plasma pharmacokinetics” refers to shortening the plasma half-life of the antibody.

  In the present invention, whether or not the plasma pharmacokinetics of the antibody is modified in a desired direction, that is, whether or not the plasma pharmacokinetics are controlled as desired is, for example, mouse, rat, rabbit, dog, monkey, etc. It can be appropriately evaluated by conducting a kinetic test using In addition, in the present invention, “extension of plasma half-life” or “reduction of plasma half-life” is more specifically, in addition to the parameter of plasma half-life (t1 / 2), mean plasma residence time It can also be grasped by any parameters such as plasma clearance (CL), area under the concentration curve (AUC), plasma half-life, etc. ("Understanding by pharmacokinetic exercise" (Nanzan-do)). For example, by performing noncompartmental analysis according to the procedure attached to the pharmacokinetic analysis software WinNonlin (Pharsight), the “control of plasma kinetics” provided by the present invention can be appropriately evaluated using these parameters.

  Moreover, it is possible to maintain the function of the antibody by controlling the pharmacokinetics in plasma. For example, if the method of the present invention is applied to an antibody having cytotoxic activity, the function can be maintained, and the function of the polypeptide before modification such as cytotoxic activity effect, antagonist activity, agonist activity, etc. can be maintained. It becomes possible to adjust the time.

  In the present invention, “amino acid residue that can be exposed on the surface” usually refers to an amino acid residue on the surface of a polypeptide constituting the antibody. “Amino acid residue on the surface of a polypeptide” means an amino acid residue whose side chain can be in contact with a solvent molecule (usually a water molecule in many cases), and all of the side chains are not necessarily solvent molecules. The amino acid residue is defined as the amino acid on the surface when even a part of the side chain contacts the solvent molecule. Those skilled in the art can create a homology model of a polypeptide or an antibody by homology modeling using commercially available software. Based on the homology model, amino acid residues on the surface of a polypeptide constituting an appropriate antibody can be suitably selected as “amino acid residues on the surface of the polypeptide”.

  In the present invention, the “amino acid residue that can be exposed on the surface” is not particularly limited, but is preferably an amino acid residue outside the FcRn binding region in the antibody. As this FcRn binding region, for example, an Fc region is preferably exemplified.

  The amino acid residue whose charge is to be altered in the antibody of the present invention is preferably an amino acid residue constituting the heavy chain variable region or the light chain variable region of the antibody. Specific examples of the variable region include a complementarity determining region (CDR) and a framework region (FR).

  A person skilled in the art can appropriately select the surface amino acid residues in the antibody variable region based on a homology model prepared by homology modeling or the like. That is, H1, H3, H5, H8, H10, H12, H13, H15, H16, H19, H23, H25, H26, H31, H39, H42, H43, which are amino acid residues based on Kabat numbering of the H chain variable region H44, H46, H61, H62, H64, H65, H68, H71, H72, H73, H75, H76, H81, H82b, H83, H85, H86, H105, H108, H110, H112 Amino acid residues can be appropriately selected. For example, in the H chain FR region of the humanized glypican 3 antibody represented by SEQ ID NO: 195, 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39 , 42, 43, 44, 46, 69, 72, 73, 74, 76, 77, 82, 85, 87, 89, 90, 107, 110, 112, 114th amino acid residue can be exemplified as surface amino acids However, the present invention is not limited to these. The surface amino acid residues in the heavy chain CDRs can also be selected by a similar homology model. That is, H97, which is an amino acid residue based on Kabat numbering, is exposed on the surface of almost all antibodies. For example, the 101st serine in the H chain CDR of the humanized anti-glypican 3 antibody represented by SEQ ID NO: 195 corresponds to the amino acid residue. As other amino acid residues in the H chain CDR of the humanized anti-glypican 3 antibody represented by SEQ ID NO: 195, amino acid residues at positions 52, 54, 62, 63, 65 and 66 are preferably mentioned.

  In the light chain variable region, L1, L3, L7, L8, L9, L11, L12, L16, L17, L18, L20, L22, L24, L27, L38, L39, L41, which are amino acid residues based on Kabat numbering, L42, L43, L45, L46, L49, L53, L54, L55, L57, L60, L63, L65, L66, L68, L69, L70, L74, L76, L77, L79, L80, L81, L85, L100, L103, A surface amino acid residue in the antibody variable region can be appropriately selected from L105, L106, and L107. For example, the humanized anti-glypican 3 antibody represented by SEQ ID NO: 195 has 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 43, 44, 45, 46, 48, Examples of surface amino acids include 49, 50, 54, 62, 65, 68, 70, 71, 73, 74, 75, 79, 81, 82, 84, 85, 86, 90, 105, 108, 110, 111, 112 However, the present invention is not limited to these. The surface amino acid residues in the L chain CDRs can also be selected by a homology model similar to the homology model by which the surface amino acid residues in the H chain CDRs are determined. As the amino acid residues in the L chain CDR of the humanized anti-glypican 3 antibody represented by SEQ ID NO: 201, the 24th, 27th, 33rd, 55th and 59th amino acid residues are preferably exemplified.

  Specifically, the “modification” of an amino acid residue in the method provided by the present invention specifically refers to substitution of the original amino acid residue with another amino acid residue, deletion of the original amino acid residue, This refers to addition of an amino acid residue, etc., but preferably refers to substitution of the original amino acid residue with another amino acid residue. That is, the “modification of charge of amino acid residue” in the present invention preferably includes amino acid substitution.

  In order to perform the above-described “modification of amino acid residue charge”, the anti-glypican 3 antibody provided by the present invention includes, for example, 19 in the heavy chain variable region constituting the humanized anti-glypican 3 antibody represented by SEQ ID NO: 195. , 43, 52, 54, 62, 63, 65, 66, 107, the charge of at least one amino acid residue is suitably modified. Further, for example, it is selected from the 17th, 24th, 27th, 33th, 55th, 59th, 79th, 82th and 105th amino acid residues in the L chain variable region constituting the humanized anti-glypican 3 antibody represented by SEQ ID NO: 201 The charge of at least one amino acid residue is suitably modified. Among the amino acid residues, amino acid residues other than the amino acid residues whose charge has been modified do not need to be modified as long as the desired plasma pharmacokinetic control effect is obtained. It may be modified as appropriate so as to have the same kind of charge as the modified amino acid residue or no charge.

  In the CDR of the anti-human IL-6 receptor antibody (6R_a_H1L1) provided by the present invention, in order to carry out the above “modification of amino acid residue charge” while maintaining the binding activity to the antigen, for example, SEQ ID NO: 221 The charge of at least one amino acid residue selected from the 31st, 64th and 65th amino acid residues is suitably modified by kabat numbering in the heavy chain variable region constituting the anti-human IL-6 receptor antibody represented . In addition, for example, at least one selected from the 24th, 27th, 53rd, and 55th amino acid residues by kabat numbering in the L chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 224 The charge of the amino acid residue is preferably modified. Among the amino acid residues, amino acid residues other than the amino acid residues whose charge has been modified do not need to be modified as long as the desired plasma pharmacokinetic control effect is obtained. It may be modified as appropriate so as to have the same kind of charge as the modified amino acid residue or no charge.

  In the CDR of the anti-human IL-6 receptor antibody (6R_b_H1L1) provided by the present invention, in order to carry out the above “modification of the charge of amino acid residues” while maintaining the binding activity to the antigen, for example, SEQ ID NO: 227 The charge of at least one amino acid residue selected from the 31st amino acid residues by kabat numbering in the heavy chain variable region constituting the anti-human IL-6 receptor antibody represented is suitably modified. In addition, for example, at least one selected from the 24th, 53rd, 54th, and 55th amino acid residues by kabat numbering in the light chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 229 The charge of the amino acid residue is preferably modified. Among the amino acid residues, amino acid residues other than the amino acid residues whose charge has been modified do not need to be modified as long as the desired plasma pharmacokinetic control effect is obtained. It may be modified as appropriate so as to have the same kind of charge as the modified amino acid residue or no charge.

  In the CDR of the anti-human GPC3 antibody provided by the present invention, for example, the anti-human GPC3 represented by SEQ ID NO: 233 is used in order to carry out the above-mentioned “modification of amino acid residue charge” while maintaining the binding activity to the antigen. The charge of at least one amino acid residue selected from the 61st, 62nd, 64th, and 65th amino acid residues by kabat numbering in the heavy chain variable region constituting the antibody is suitably modified. In addition, for example, the charge of at least one amino acid residue selected from the 24th and 27th amino acid residues by kabat numbering in the L chain variable region constituting the anti-human GPC3 antibody represented by SEQ ID NO: 236 is It is suitably modified. Among the amino acid residues, amino acid residues other than the amino acid residues whose charge has been modified do not need to be modified as long as the desired plasma pharmacokinetic control effect is obtained. It may be modified as appropriate so as to have the same kind of charge as the modified amino acid residue or no charge.

  The CDR of the anti-human IL-31 receptor antibody provided by the present invention is represented by, for example, SEQ ID NO: 239 in order to carry out the above “modification of the charge of amino acid residue” while maintaining the binding activity to the antigen. The charge of at least one amino acid residue selected from the 61st, 62nd, 64th, and 65th amino acid residues by kabat numbering in the heavy chain variable region constituting the anti-human IL-31 receptor antibody is suitably modified. In addition, for example, at least one amino acid residue selected from the 24th and 54th amino acid residues by kabat numbering in the light chain variable region constituting the anti-human IL-31 receptor antibody represented by SEQ ID NO: 242 The charge is suitably modified. Among the amino acid residues, amino acid residues other than the amino acid residues whose charge has been modified do not need to be modified as long as the desired plasma pharmacokinetic control effect is obtained. It may be modified as appropriate so as to have the same kind of charge as the modified amino acid residue or no charge.

  Among amino acids, it is known that there are charged amino acids. Generally, lysine (K), arginine (R), and histidine (H) are known as positively charged amino acids (positively charged amino acids). As the negatively charged amino acid (negatively charged amino acid), aspartic acid (D), glutamic acid (E) and the like are known. Other amino acids are known as non-charged amino acids.

The “modified amino acid residue” is preferably appropriately selected from the amino acid residues included in any of the following groups (a) or (b), but is not particularly limited to these amino acids.
(A) Glutamic acid (E), aspartic acid (D)
(B) Lysine (K), Arginine (R), Histidine (H)

  In addition, when the original amino acid residue (before modification) has already been charged, it is also a preferred embodiment of the present invention to modify the amino acid residue to have no charge. That is, the modification in the present invention includes (1) substitution of an amino acid having a charge with an amino acid having no charge, (2) substitution of an amino acid having a charge with an amino acid having a charge opposite to that of the amino acid, 3) Substitution of an amino acid having no charge to an amino acid having a charge.

  In the present invention, it is preferable that amino acid residues constituting the antibody are modified so that the isoelectric point (pI) of the antibody changes. In addition, when there are a plurality of amino acid residues to be modified, the amino acid residues subjected to modification may contain a small number of amino acid residues having no charge.

  Preferable examples of “modification of amino acid residue charge” in the anti-glypican 3 antibody provided by the present invention include the following. As the modification that increases the pI value, for example, at least one substitution of Q43K, D52N, and Q107R in the heavy chain variable region constituting the humanized anti-glypican 3 antibody represented by SEQ ID NO: 195 can be performed. Preferably, the amino acid sequence is represented by SEQ ID NO: 198. Further, for example, at least one substitution of E17Q, Q27R, Q105R in the L chain variable region constituting the humanized anti-glypican 3 antibody represented by SEQ ID NO: 201 can be performed, and particularly preferably, SEQ ID NO: 204 Modified to the amino acid sequence shown. On the other hand, as a modification that decreases the pI value, substitution of at least one of K19T, Q43E, K63S, K65Q, and G66D in the heavy chain variable region constituting the humanized anti-glypican 3 antibody represented by SEQ ID NO: 195 is performed. The amino acid sequence represented by SEQ ID NO: 197 is particularly preferably modified. In addition, for example, at least one substitution of Q27E, K79T, and R82S in the L chain variable region constituting the humanized anti-glypican 3 antibody represented by SEQ ID NO: 201 can be performed, and particularly preferably, SEQ ID NO: 203 Modified to the amino acid sequence shown.

  Preferable examples of the “modification of amino acid residue charge” in the anti-human IL-6 receptor antibody (6R_a_H1L1) provided by the present invention include at least one amino acid substitution among the amino acid substitutions described in Table 20.

  Preferable examples of “modification of charge of amino acid residue” in the anti-human IL-6 receptor antibody (6R_b_H1L1) provided by the present invention include at least one amino acid substitution among the amino acid substitutions described in Table 22.

  Preferable examples of “modification of amino acid residue charge” in the anti-human GPC3 antibody provided by the present invention include at least one amino acid substitution described in Table 24.

  Preferable examples of “modification of charge of amino acid residue” in the anti-human IL-31 receptor antibody provided by the present invention include at least one amino acid substitution among the amino acid substitutions described in Table 27.

  In the present invention, the number of amino acid residues subjected to the modification is not particularly limited. For example, when the variable region of an antibody is modified, the binding activity with an antigen is not decreased and the immunogenicity is not increased. In addition, it is preferred that the minimum amino acid residues necessary to achieve the desired controlled plasma pharmacokinetics are modified. In addition, an appropriate combination of an amino acid residue modification that causes an increase in binding activity with an antigen and an amino acid residue modification that causes a decrease in immunogenicity can also be suitably performed.

  A known means can be used for measuring the antigen binding activity of the antibody. For example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent immunoassay can be used. The above method is described in a general teaching document "Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988".

  Examples of the method for measuring the binding activity of an antibody to cells include the method described on pages 359-420 in Antibodies A Laboratory Manual. (Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). That is, it can be evaluated by the principle of BIACORE using cells as an antigen, cell proliferation assay, ELISA or FACS (fluorescence activated cell sorting). In the ELISA format, the binding activity of an antibody to a cell is quantitatively evaluated by comparing the signal level generated by the enzymatic reaction. That is, a test antibody is added to an ELISA plate on which each forced expression cell is immobilized, and an antibody bound to the cell is detected using an enzyme-labeled antibody that recognizes the test antibody. Alternatively, in FACS, the binding activity to cells can be compared by preparing a dilution series of test antibodies and determining the antibody binding titer (titer) for each forcedly expressed cell.

Binding between an antigen that is not bound to a carrier such as an ELISA plate and is expressed on the surface of a cell suspended in a buffer or the like and an antibody against the antigen can be measured by the FACS format. Examples of flow cytometers used for such measurements include FACSCanto ^™ II, FACSAria ^™ , FACSArray ^™ , FACSVantage ^™ SE, FACSCalibur ^™ (BD Biosciences), EPICS ALTRA HyPerSort, Cytomics FC 500, EPICS XL- Examples include MCL ADC, EPICS XL ADC, and Cell Lab Quanta / Cell Lab Quanta SC (Beckman Coulter).

  As an example of a preferable method for measuring the binding activity of an antibody to an antigen, a cell expressing the antigen is reacted with a test antibody, the cell is stained with a FITC-labeled secondary antibody that recognizes the test antibody, and then FACSCalibur ( BD), and the fluorescence intensity can be analyzed using CELL QUEST Software (BD). According to this method, after staining with a FITC-labeled secondary antibody that specifically recognizes a test antibody bound to an antigen on the cell surface that expresses the antigen, the fluorescence is measured when measured by FACSCalibur. It can be judged by comparing the Geometric Mean value (test Geo-Mean value) obtained by the method of analyzing the intensity using CELL QUEST Software with the control Geo-Mean value obtained using the control antibody. . The calculation formula for obtaining the Geo-Mean value (Geometric Mean) is described in CELLQUEST Software User's Guide (BD biosciences).

  Further, in order not to increase the immunogenicity in the human body to which the antibody is administered, the modified amino acid sequence is preferably a human sequence (a sequence found in natural antibodies derived from humans). It is not limited to. Furthermore, a mutation can be suitably introduced at a site other than the alteration introduced so that the isoelectric point changes so that each of a plurality of FRs (FR1, FR2, FR3, FR4) becomes a human sequence after the alteration. The method for replacing each FR with a human sequence in this way is described in non-patent literature (Ono K, Ohtomo T, Yoshida K, Yoshimura Y, Kawai S, Koishihara Y, Ozaki S, Kosaka M, Tsuchiya M., The humanized anti-HM1. .24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity., Mol Immunol. (1999) 36 (6), 387-395). In addition, in order to change the isoelectric point of the antibody, it may be modified to another human FR so that the charge of each FR is changed (for example, FR3 is changed from another human FR so that the isoelectric point of the antibody is lowered). May be replaced). Such humanization methods are described in non-patent literature (Dall'Acqua WF, Damschroder MM, Zhang J, Woods RM, Widjaja L, Yu J, Wu H .., Antibody humanization by framework shuffling., Methods. (2005) 36 ( 1), 43-60).

  In addition, when the target controlled plasma pharmacokinetics is not reached by a slight modification of the surface charge, the target controlled plasma is repeatedly performed by repeatedly modifying the surface charge and evaluating the plasma pharmacokinetics. Desired antibodies exhibiting moderate pharmacokinetics can be suitably obtained.

  Non-patent literature (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL.Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P- selectin. J Immunol. (1998), 160 (2), 1029-35), chimera EP5C7.g4, which is a chimeric antibody (IgG4) of anti-E, P-Selectin antibody, and HuEP5C7, which is a humanized antibody (IgG4) Comparison of plasma pharmacokinetics in .g4 rhesus monkeys has been shown to be comparable. Non-patent literature (Gobburu JV, Tenhoor C, Rogge MC, Frazier DE Jr, Thomas D, Benjamin C, Hess DM, Jusko WJ. Pharmacokinetics / dynamics of 5c8, a monoclonal antibody to CD154 (CD40 ligand) suppression of an immune response in monkeys. J Pharmacol Exp Ther. (1998) 286 (2), 925-30), comparing the pharmacokinetics in plasma of cynomolgus monkeys of anti-CD154 chimeric antibody ch5d8 and humanized antibody Hu5c8. It has been shown that the plasma pharmacokinetics of both are equivalent. Non-patent literature (Kashmiri SV, Shu L, Padlan EA, Milenic DE, Schlom J, Hand PH., Generation, characterization, and in vivo studies of humanized anticarcinoma antibody CC49., Hybridoma. (1995) 14 (5), 461- 73) shows that the plasma pharmacokinetics of the chimeric antibody cCC49 and the humanized antibody HuCC49 in mice are equivalent. Non-patent literature (Graves SS, Goshorn SC, Stone DM, Axworthy DB, Reno JM, Bottino B, Searle S, Henry A, Pedersen J, Rees AR, Libby RT., Molecular modeling and preclinical evaluation of the humanized NR-LU -13 antibody., Clin Cancer Res. (1999) 5 (4), 899-908) and non-patent literature (Couto JR, Blank EW, Peterson JA, Ceriani RL., Anti-BA46 monoclonal antibody Mc3: humanization using a novel In positional consensus and in vivo and in vitro characterization., Cancer Res. (1995) 55 (8), 1717-22), in mouse evaluation, the plasma pharmacokinetics and distribution of mouse antibody and humanized antibody are equivalent. Since both mouse Fc and human Fc cross mouse FcRn, the plasma pharmacokinetics and distribution of the chimeric antibody and the humanized antibody are considered to be equivalent. As shown in these examples, plasma pharmacokinetics are comparable between chimeric and humanized antibodies with the same CDR. That is, non-patent literature (Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. (1997) 15 (7), 637-40) and the like, the pharmacokinetics in plasma are equivalent to those of chimeric antibodies when humanized by known methods, and humans whose plasma pharmacokinetics are controlled by known methods An antibody cannot be produced.

In contrast, when the method found in the present invention is used, when the chimeric antibody is humanized, the amino acid residues that can be exposed on the surface of the chimeric antibody are modified to modify the pI of the antibody. A humanized antibody with controlled plasma pharmacokinetics compared to a chimeric antibody (ie, having an extended plasma half-life or a reduced plasma half-life) can be produced. Modifications of amino acids that can be exposed on the surface of a humanized antibody to control plasma pharmacokinetics may be performed simultaneously with the humanization of the antibody, or may be exposed on the surface using the humanized antibody as a starting material. The pI of the humanized antibody may be further modified by modifying amino acid residues that can be obtained.
The value of the isoelectric point in the present invention can be measured by isoelectric focusing known to those skilled in the art. The value of theoretical isoelectric point can be calculated using gene and amino acid sequence analysis software (Genetyx etc.). In the present invention, it is useful when it is necessary to change the isoelectric point significantly, for example, for sufficient control of plasma pharmacokinetics. In particular, it is preferable to change the value of the isoelectric point by 1.0 or more as the theoretical isoelectric point value, and more preferable when the value of 3.0 or more is required.

  Non-patent literature (Adams CW, Allison DE, Flagella K, Presta L, Clarke J, Dybdal N, McKeever K, Sliwkowski MX. Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab., Cancer Immunol Immunother. 2006) 55 (6), 717-27), the plasma pharmacokinetics of the three humanized antibodies trastuzumab, bevacizumab, and pertuzumab, which were humanized using the same human antibody FR sequence, were almost the same. Is marked. That is, when humanization is performed using the same FR sequence, plasma pharmacokinetics are almost the same. According to the method found in the present invention, in addition to the humanization step as described above, by modifying the amino acid residue that can be exposed on the surface of the antibody to modify the pI of the antibody, It is possible to control the plasma concentration.

  The method of the present invention can also be applied to human antibodies. The amino acid residue that can be exposed on the surface of a human antibody prepared from a human antibody library or a human antibody-producing mouse is modified, and the pI of the human antibody is modified to modify the first human antibody. It is possible to produce human antibodies whose plasma pharmacokinetics are controlled compared to plasma pharmacokinetics (ie, whose plasma half-life is increased or whose plasma pharmacokinetics are shortened).

  Decreasing the pI value of an antibody extends the plasma half-life of the antibody. On the other hand, it is known that increasing the pI value of the antibody shortens the plasma half-life and improves the tissue migration of the antibody (Vaisitti T, Deaglio S, Malavasi F., Cationization of monoclonal). antibodies: another step towards the "magic bullet" ?, J Biol Regul HomeostAgents. (2005) 19 (3-4), 105-12, Pardridge WM, Buciak J, Yang J, Wu D. Enhanced endocytosis in cultured human breast carcinoma cells and in vivo biodistribution in rats of a humanized monoclonal antibody after cationization of the protein. (1998) 286 (1), 548-54). However, since the antibody has increased immunogenicity and increased internalization activity in the cell, the internalization activity in the cell is an inhibitory factor for the exertion of ADCC and CDC activity. In order to apply to an antibody having an effect on cancer treatment through a mechanism such as cytotoxic activity, further improvement has been demanded. In other words, for antibodies that exert an effect on cancer treatment through mechanisms such as cytotoxic activity in which the internalization activity into cells is an inhibitory factor in exerting such activities such as ADCC and CDC activity, either increase or decrease in pI value It was not known whether would provide an enhanced tumor suppressive effect. In the present invention, a humanized antibody modified antibody having a decreased pI value and a humanized antibody modified antibody having an increased pI value are prepared, and the antitumor effects of the two are compared, whereby any of the modified tumors is high. It has been verified whether it has an inhibitory effect. As a result, it was surprisingly shown that a humanized antibody having a reduced pI value exerts a better effect on liver cancer.

  As the “antibody” in the present invention, substitution, deletion, addition and / or insertion of amino acid residues constituting the antibody, etc. using an antibody in which the charge of amino acid residues is modified as described above as a starting material. Includes an antibody whose amino acid sequence is further modified. In addition, the “antibody” in the present invention uses, as a starting material, an antibody whose amino acid sequence has been modified by amino acid residue substitution, deletion, addition and / or insertion, chimerization or humanization, and the like. Antibodies in which the charge of amino acid residues constituting the antibody is further modified are also included.

  As an example of the modification aimed at improving the properties of the antibody provided by the present invention, a modification aimed at enhancing the stability of the antibody (hereinafter referred to as stability modification) is preferably mentioned. Antibodies in aqueous solution are equilibrated between two states, the native state and the inactive denatured state. The stability of the natural state is expressed by the second law of thermodynamics (ΔG = ΔH-TΔS). And entropy change ΔS (due to changes in solvation and three-dimensional freedom). A positive value ΔG means that the native state of the protein is more stable than the denatured state of the protein, and when ΔG shows a larger positive value, the stability of the native state of the protein is further increased. In order to denature proteins, it is necessary to break the force that contributes to this stabilization. For example, subjecting a protein solution to high temperatures increases the degree of freedom of the three-dimensional structure and weakens factors that contribute to protein stabilization, leading to thermal denaturation of the protein. In this case, the -TΔS term governs denaturation. Will do. The ΔH of unfolding due to protein thermal denaturation can be measured directly using DSC (differential scanning calorimetry) as specifically described in the examples described herein. . The DSC curve in the heat denaturation process of a protein becomes an endothermic peak across a temperature that is specific to the test protein called the midpoint of denaturation (Tm). The denaturation enthalpy change is obtained by integrating the peak. In general, the value of Tm is one index of thermal stability. The change in heat capacity (ΔCp) when the protein undergoes heat denaturation by DSC can also be measured. The heat capacity change that accompanies thermal denaturation is primarily due to the hydration of amino acid residues that are not exposed on the surface of the molecule when the protein is present in the natural state and exposed to solvent molecules as the protein is denatured. caused by.

  As described above, the “modification” of an amino acid residue in the method provided by the present invention specifically means substitution of the original amino acid residue with another amino acid residue, or deletion of the original amino acid residue. , Adding a new amino acid residue, etc., preferably, substituting the original amino acid residue with another amino acid residue. That is, for modification of antibody stability in the present invention, modification by amino acid substitution is preferably used. As a result of the stability modification to the amino acid residues constituting the antibody, the Tm value of the antibody increases. That is, the Tm value is preferably used as an index to which antibody stability has been altered.

  In order to perform the above-mentioned “stability modification”, the anti-glypican 3 antibody provided by the present invention is, for example, 37, 40, At least one amino acid residue selected from the 48th and 51st amino acid residues is preferably modified. In addition, for example, at least 1 selected from amino acid residues 2, 25, 42, 48, 50, 83, 84 in the L chain variable region constituting the humanized anti-glypican 3 antibody represented by SEQ ID NO: 201 One amino acid residue is suitably modified. Among the amino acid residues described above, amino acid residues other than the amino acid residues subjected to the stability modification do not need to be modified as long as a desired Tm value is obtained. The humanized anti-glypican 3 antibody can be appropriately modified so as to have a Tm value equal to or higher than the Tm value.

  The stability modification can be carried out by randomly modifying each amino acid residue constituting the humanized antibody to be subjected to modification. Further, a part of the amino acid sequence constituting the humanized antibody to be subjected to the modification is an amino acid sequence constituting an existing antibody having a high Tm value, and a part of the amino acid sequence of the humanized antibody to be subjected to the modification It can also be carried out by substituting the corresponding sequence from the viewpoint of the three-dimensional structure correlation of the antibody. The position of the amino acid residue to be substituted is not limited, but the amino acid residue in the FR region can be suitably modified. In addition, even amino acid residues in the CDR region can be appropriately modified as long as they do not involve a reduction in antigen binding activity. In addition, the number of amino acid residues to be modified is not particularly limited, and the amino acid residue can be changed by substituting a specific segment of the FR region with a desired segment. In the FR region, all of the FR1, FR2, FR3, and FR4 segments in the FR region can be modified, or can be implemented by a combination of one or more modified segments.

  In the case of modifying a segment of the FR region, a FR2 region of H chain or L chain can be mentioned as a suitable example. For example, the modification of the amino acid residue that modifies the FR2 of the H chain of the humanized glypican 3 antibody having the VH1b subclass represented by SEQ ID NO: 195 to the VH4 subclass, that is, V37I that replaces the 37th valine with isoleucine Similarly, modifications of A40P, M48I, and L51I are mentioned as preferred specific examples. Also, for example, modification of the L chain FR2 region of the humanized anti-glypican 3 antibody having the VK2 subclass represented by SEQ ID NO: 201 to the VK3 subclass, that is, modification of L42Q, S48A, Q50R, and further reproduction of FR1 A suitable specific example is a modification of V2I corresponding to a modification to a cell lineage sequence.

  Any substitution of amino acid residues constituting the antibody, deletion, addition and / or insertion, and modification of the amino acid sequence such as humanization and chimerization can be suitably performed by methods known to those skilled in the art. Similarly, when the antibody provided by the present invention is produced as a recombinant antibody, substitution, deletion, addition and / or insertion of amino acid residues constituting the variable region and constant region of the antibody can be suitably performed.

  As the antibody in the present invention, any animal-derived antibody such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody is preferably used. Furthermore, for example, a modified antibody in which the amino acid sequence is substituted, such as a chimeric antibody, particularly a humanized antibody, can be preferably used. In addition, modified antibodies to which various molecules are bound can also be suitably used.

  A “chimeric antibody” is an antibody produced by combining sequences derived from different animals. For example, an antibody composed of mouse antibody H chain and L chain variable (V) regions and human antibody H chain and L chain constant (C) regions can be suitably exemplified. Methods for producing chimeric antibodies are known. For example, a recombinant DNA in which a DNA encoding the antibody V region and a DNA encoding the human antibody C region are fused in frame is incorporated into a commonly used expression vector. By culturing a host cell into which the vector has been introduced, a chimeric antibody can be appropriately obtained or isolated from the culture solution.

  Humanized antibodies are also referred to as reshaped human antibodies, and are complementary determining regions (CDRs) of antibodies isolated from mammals other than humans, such as mice, and framework regions of human antibodies ( FR; framework region). A DNA sequence encoding a humanized antibody can be synthesized by an overlap PCR reaction using a plurality of oligonucleotides as templates. The material for the overlapping PCR reaction and the method for carrying out the same are described in WO98 / 13388 and the like. The DNA encoding the variable region of the humanized antibody of the present invention is obtained by overlapping PCR from a plurality of oligonucleotides prepared so as to have nucleotide sequences overlapping each other, which is a DNA encoding a human antibody constant region And so that a codon sequence is formed in frame. The DNA ligated as described above is then inserted into an expression vector so that the DNA is expressed and introduced into the host.

  Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md .; Chothiaet al., Nature (1989) 342, 877). In addition, general gene recombination techniques are also known (see European Patent Application Publication No. EP 125023, WO 96/02576). By using these known methods, for example, after the CDR of an antibody obtained from a non-human animal such as a mouse antibody is determined, a recombinant antibody in which the CDR and the FR of a human antibody are linked is encoded. DNA is constructed. The FR of the human antibody linked via CDR is selected so that the CDR forms a good antigen binding site. If necessary, FR amino acid residues in the variable region of the antibody can be modified as appropriate so that the CDRs of the reshaped human antibody form an appropriate antigen binding site (Sato et al., Cancer Res. (1993) 53, 851-6). The amino acid residues in the FR that are subject to modification include residues that directly bind to the antigen by non-covalent bonds (Amit et al., Science (1986) 233, 747-53), and residues that affect or act on the CDR structure. Groups (Chothia et al., J. Mol. Biol. (1987) 196, 901-17) and residues related to VH-VL interaction (EP239400 patent publication) are included.

  A humanized antibody encoded by the DNA produced by a host cell transformed or transduced by a commonly used expression vector into which the DNA has been inserted can be isolated from the culture by culturing the host cell. Be released.

  When the antibody provided by the present invention is an antibody, humanized antibody or human antibody, a C region derived from a human antibody is preferably used as the C region of the antibody. For example, Cγ1, Cγ2, Cγ3, and Cγ4 can be suitably used as the H chain C region, and Cκ and Cλ can be suitably used as the L chain C region. In addition, the human antibody C region can be modified as appropriate in order to improve the stability of the antibody or its production. The chimeric antibody provided by the present invention is suitably composed of the V region of an antibody obtained from a mammal other than a human and the C region of a human antibody. On the other hand, a humanized antibody is preferably composed of an CDR of an antibody obtained from a mammal other than a human and the FR and C regions of a human antibody. In addition, the human antibody is preferably composed of an antibody CDR obtained from a human and the FR and C regions of the human antibody. The C region of a human antibody is composed of a unique amino acid sequence corresponding to an isotype such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. As the C region of the humanized antibody provided by the present invention, the C region of an antibody belonging to any isotype is preferably used. Preferably, the C region of human IgG is used, but is not limited thereto. Further, the FR of a human antibody used as a humanized antibody or FR of a human antibody is not particularly limited, and an FR of an antibody belonging to any isotype is also preferably used.

  Non-patent literature (Ono K, Ohtomo T, Yoshida K, Yoshimura Y, Kawai S, Koishihara Y, Ozaki S, Kosaka M, Tsuchiya M., The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity., Mol Immunol. (1999) 36 (6), 387-395). Substitution in whole or in part with germline sequences can also be applied. Based on a reasonable prediction that germline sequences will be less immunogenic, the amino acid sequences that make up the FR regions of humanized antibodies are compared by aligning them with germline amino acid sequences (Abhinandan KR and Martin CR, J. Mol. Biol., (2007) 369, 852-862). As long as the binding activity to the antigen is not lost, amino acid residues constituting FR regions of different humanized antibodies in the comparison can be substituted with amino acid residues in germline sequences. As a specific example, among the amino acid residues constituting the heavy chain variable region represented by SEQ ID NO: 195, the 70th L is I, the 87th T is R, the 97th T is A Modifications to be substituted are included. Moreover, the modification etc. which substitute 25th S by A among the amino acid residues which comprise the L chain variable region represented by sequence number: 201 are mentioned.

  The modified chimeric antibody, humanized antibody, and human antibody variable region and constant region provided by the present invention constitute a variable region and a constant region of the antibody subjected to the modification as long as they exhibit binding specificity to the antigen. Alternatively, deletion, substitution, insertion and / or addition of more amino acids can be suitably performed.

  Chimeric antibodies, humanized antibodies and human antibodies using human-derived sequences are useful when used as therapeutic antibodies to be administered to humans for therapeutic purposes, etc., because their immunogenicity in the human body is reduced. Conceivable.

  As the sequence of the gene encoding the H chain or L chain of the antibody before mutagenesis in the method of the present invention, a known sequence can be used. A simple array can be obtained. The gene can be suitably obtained from an antibody library, for example. Furthermore, the gene can also be obtained by cloning using a known technique such as RT-PCR using a hybridoma mRNA producing a monoclonal antibody as a template.

  Many antibody libraries are already known as antibody libraries. Moreover, since the preparation method of an antibody library is also well-known, those skilled in the art can obtain or produce an antibody library suitably. For example, suitable antibody libraries include Clackson et al., Nature (1991) 352, 624-8, Marks et al., J. Mol. Biol. (1991) 222, 581-97, Waterhouseset al., Nucleic Acids. Res. (1993) 21, 2265-6, Griffiths et al., EMBO J. (1994) 13, 3245-60, Vaughan et al., Nature Biotechnology (1996) 14, 309-14, and JP 20- The antibody phage library disclosed by literatures, such as 504970 gazette, is illustrated. In addition, known methods such as a method for preparing a library in eukaryotic cells (WO95 / 15393 pamphlet) and a ribosome display method are preferably used. Furthermore, techniques for obtaining human antibodies by panning using a human antibody library as a starting material are known to those skilled in the art. That is, a single chain antibody (scFv) in which the variable regions of the H chain and L chain of a human antibody are fused in frame is expressed on the surface of the phage by the phage display method. By selecting phage that bind to the antigen, the gene encoding the scFv that binds to the antigen is isolated from the phage. By identifying the sequence of the gene, the sequence of the DNA encoding the variable region of the heavy and light chains of the antibody that binds to the antigen can be determined. An antibody gene having the sequence is appropriately inserted into an expression vector and expressed in an appropriate host cell as described later, whereby a human antibody is appropriately obtained. These methods are already well known, and the methods disclosed in WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388 are exemplified.

  As a method for obtaining a gene encoding an antibody from a hybridoma producing a monoclonal antibody, basically known techniques can be employed. As will be described in detail below, in brief, after an animal is immunized with a desired sensitizing antigen according to a normal immunization method, immune cells obtained from the animal are mixed with known parental cells by a normal cell fusion method. Donated for cell fusion. According to the usual screening method, monoclonal antibody-producing cells (hybridoma) are screened, and cDNA of the variable region (V region) of the antibody is synthesized by reverse transcriptase using mRNA obtained from the selected hybridoma as a template. The An antibody gene is suitably obtained by fusing the cDNA in-frame with a DNA encoding a desired antibody constant region (C region).

  More specifically, the following examples are preferably exemplified, but the present invention is not limited to these examples. The sensitizing antigen used to obtain the antibody provided by the present invention may be a complete antigen having immunogenicity or an incomplete antigen including a hapten that does not exhibit immunogenicity. Good. For example, a full-length protein or a partial polypeptide or peptide thereof can be preferably used. A preferred specific example is the soluble GPC3 core polypeptide represented by SEQ ID NO: 207. In addition, substances composed of polysaccharides, nucleic acids, lipids and the like are also known to act as antigens, and the antigen to which the antibody of the present invention binds is not particularly limited to the above-described substance modes. The preparation of the antigen is preferably performed by a method known to those skilled in the art, and for example, a method using baculovirus (for example, WO98 / 46777 etc.) can be preferably used. When the immunogenicity of the antigen is low, the animal can be suitably immunized with the antigen bound to an immunogenic macromolecule such as albumin. When the sensitizing antigen is a molecule that penetrates the cell membrane, if necessary, a polypeptide fragment in the extracellular region of the molecule is suitably used as the sensitizing antigen. Alternatively, cells that express the molecule on the cell surface can be suitably used as the sensitizing antigen. Further, when the sensitizing antigen is an insoluble molecule, the molecule is solubilized by binding to another water-soluble molecule, and the solubilized binding molecule is suitably used as the sensitizing antigen.

  Antibody-producing cells are preferably obtained by immunizing an animal with the appropriate sensitizing antigen. Alternatively, antibody-producing cells can be obtained by immunizing in vitro lymphocytes capable of producing antibodies. As immunized animals, various vertebrates and mammals can be used. In particular, rodents, rabbits, and primates are generally used as animals to be immunized. Examples include rodents such as mice, rats and hamsters, rabbits such as rabbits, and primates such as monkeys such as cynomolgus monkeys, rhesus monkeys, baboons and chimpanzees. In addition, transgenic animals that retain a repertoire of human antibody genes on their genome are also known, and human antibodies can be suitably obtained by using such animals (WO96 / 34096; Mendez et al., Nat Genet. (1997) 15, 146-56). Instead of using such transgenic animals, for example, human lymphocytes are sensitized with the desired antigen or cells expressing the desired antigen in vitro and then fused with human myeloma cells such as U266. Thus, a desired human antibody having binding activity to the antigen can be suitably obtained (see Japanese Patent Publication No. 1-59878). In addition, a transgenic animal carrying all repertoires of human antibody genes on its genome is immunized with a desired antigen (see WO93 / 12227, WO92 / 03918, WO94 / 02602, WO96 / 34096, WO96 / 33735) ) The desired human antibody can be suitably obtained.

  For immunization of animals, for example, a sensitizing antigen is appropriately diluted and suspended in Phosphate-Buffered Saline (PBS) or physiological saline, etc., and if necessary emulsified by mixing with an adjuvant, then the sensitizing antigen. Is performed by intraperitoneal or subcutaneous injection of the animal. Thereafter, preferably a sensitizing antigen mixed with Freund's incomplete adjuvant is administered several times every 4 to 21 days. Confirmation of the production of antibodies against the sensitized antigen in the immunized animal can be carried out by determining the antibody titer in the animal's serum against the sensitized antigen by conventional methods such as enzyme-linked immunosorbent assay (ELISA), flow cytometry ( FACS) and the like can be carried out by measuring by a known analysis method.

  A hybridoma fuses an antibody-producing cell obtained from an animal or lymphocyte immunized with a desired sensitizing antigen with a myeloma cell using a fusion agent (for example, polyethylene glycol) commonly used for cell fusion. (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) 59-103). The hybridoma can be suitably produced according to, for example, the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. (1981) 73, 3-46). By culturing and proliferating the hybridoma cells produced by the above method, a monoclonal antibody that specifically binds to the antigen protein produced by the hybridoma is obtained. The binding specificity of the monoclonal antibody to the antigen protein can be appropriately measured by a known analysis method such as immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS). Thereafter, if necessary, the hybridoma producing the antibody whose desired specificity, binding activity or activity is measured is preferably subcloned by a technique such as limiting dilution, and the monoclonal antibody produced by the hybridoma is isolated. Can be done.

  Subsequently, a gene encoding the selected antibody is complementary to a probe (for example, a sequence encoding an antibody constant region) that can specifically bind to the gene from the hybridoma or antibody-producing cells (sensitized lymphocytes, etc.) described above. A typical oligonucleotide etc.). Alternatively, it can be cloned by RT-PCR using mRNA obtained from a hybridoma or antibody-producing cells (such as sensitized lymphocytes) as a template. Immunoglobulins are classified into five different classes based on their structural and functional differences: IgA, IgD, IgE, IgG and IgM. Furthermore, each class is classified into several isotypes (eg, IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2, etc.). Antibodies provided by the present invention may be derived from antibodies belonging to any of these classes and subclasses, and are not particularly limited to any class and subclass, but antibodies belonging to the IgG class include Particularly preferred.

  The gene encoding the amino acid sequence constituting the H chain and L chain of the antibody can be appropriately modified by genetic engineering techniques. For example, by altering nucleic acid residues that encode amino acid sequences constituting antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheep antibodies, camel antibodies, etc. A genetically engineered antibody having a target artificial modification, such as a chimeric antibody or a humanized antibody, can be appropriately produced. A chimeric antibody is an antibody composed of the H chain and L chain variable regions of a mammal other than a human, for example, a mouse, and the H chain and L chain constant regions of a human antibody, for example, derived from a mouse. DNA encoding the variable region of the antibody to be ligated to DNA encoding the constant region of the human antibody, and a recombinant vector in which this is incorporated into an expression vector is introduced into the host and expressed. Humanized antibodies, also called reshaped human antibodies, are complementary determining regions (CDRs) of antibodies isolated from mammals other than humans such as mice and the framework of human antibodies. It is an antibody linked to a region so that a codon sequence is formed in frame. The DNA sequence encoding this humanized antibody can be synthesized by an overlap PCR reaction using a plurality of oligonucleotides as templates. The material for the overlapping PCR reaction and the method for carrying out the same are described in WO98 / 13388 and the like.

  The DNA encoding the variable region of the recombinant antibody of the present invention is obtained by overlapping PCR from a plurality of oligonucleotides prepared so as to have overlapping nucleotide sequences, which encodes a human antibody constant region. Ligated to form a codon sequence in-frame with DNA. The DNA ligated as described above is then inserted into an expression vector so that the DNA is expressed and introduced into the host. The antibody encoded by the DNA is expressed by culturing the host. The expressed antibody can be obtained by appropriately purifying from the culture medium or the like of the host (see EP239400; WO96 / 02576). The FR of the humanized antibody linked via CDR is selected such that the complementarity determining region forms a good antigen binding site for the antigen. If necessary, the complementarity-determining region of the reshaped human antibody can be modified by appropriately substituting the amino acid residues constituting the FR of the variable region of the selected antibody so as to form an appropriate antigen-binding site for the antigen. (K. Sato et al., Cancer Res. (1993) 53, 851-856).

  In addition to the above-described modifications related to humanization, modifications for improving the biological properties of the antibody such as the binding activity with the antigen recognized by the antibody can be performed. The modification in the present invention can be suitably performed by methods such as site-specific mutation (see, for example, Kunkel, Proc. Natl. Acad. Sci. USA (1985) 82, 488), PCR mutation, cassette mutation and the like. In general, the amino acid sequence constituting a modified antibody having improved biological properties is 70% or more, more preferably 80% or more, and still more preferably 90% or more (eg, 95% or more, 97%, 98%, 99%) and the like and / or similarity to the amino acid sequences that make up the antibody subjected to modification (ie, the antibody on which the modified antibody is based). In the present specification, sequence identity and / or similarity refers to an antibody on which a modified antibody is based after aligning sequences and introducing gaps as necessary so that the sequence identity takes the maximum value. The percentage of amino acid residues that are identical (same residues) or similar (amino acid residues classified into the same group based on the characteristics of the side chains of general amino acids). Natural amino acid residues are usually based on their side chain properties (1) hydrophobicity: alanine, isoleucine, valine, methionine and leucine; (2) neutral hydrophilicity: asparagine, glutamine, cysteine, threonine and serine. (3) acidity: aspartic acid and glutamic acid; (4) basicity: arginine, histidine and lysine; (5) residues affecting chain orientation: glycine and proline; and (6) aromaticity: tyrosine, tryptophan. And phenylalanine.

  Further, as a modification for the purpose of enhancing the function of the antibody, for example, an improvement of the cytotoxic activity exhibited by an antibody such as a humanized antibody is preferably mentioned as a specific embodiment. Preferred examples of the cytotoxic activity include antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and the like. Can be done. In the present invention, CDC activity refers to cytotoxic activity by the complement system. On the other hand, ADCC activity means that when a specific antibody is attached to the cell surface antigen of the target cell, Fcγ receptor-bearing cells (immune cells, etc.) bind to the Fc portion via the Fcγ receptor, causing damage to the target cell. Refers to activity. Whether a test antibody has ADCC activity or CDC activity can be measured by a known method (for example, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al John Wiley & Sons, Inc., (1993) etc.).

Specifically, first, effector cells, complement solution, and target cells are prepared.
(1) Preparation of effector cells
The spleen is removed from a CBA / N mouse or the like, and the spleen cells are isolated in RPMI1640 medium (Invitrogen). After washing with the same medium containing 10% fetal bovine serum (FBS, HyClone), effector cells can be prepared by adjusting the cell concentration to 5 × 10 ⁶ cells / ml.
(2) Preparation of complement solution
A complement solution can be prepared by diluting Baby Rabbit Complement (CEDARLANE) 10-fold in a medium containing 10% FBS (Invitrogen).
(3) Preparation of target cells For 1 hour at 37 ° C in 10% FBS-containing DMEM medium with 0.2 mCi of 51Cr-sodium chromate (GE Healthcare Bioscience) The target cells can be radiolabeled by culturing. Cells expressing the antigen protein to which the test antibody binds include cells transformed with a gene encoding the antigen protein to which the test antibody binds, ovarian cancer, prostate cancer, breast cancer, uterine cancer, liver cancer, lung cancer, pancreatic cancer, Gastric cancer, bladder cancer, colon cancer cells and the like can be used. After radiolabeling, the target cells can be prepared by washing the cells three times with RPMI 1640 medium containing 10% FBS and adjusting the cell concentration to 2 × 10 ⁵ cells / ml.

  ADCC activity or CDC activity can be measured by the method described below. In the case of measuring ADCC activity, 50 μl each of target cells and test antibody are added to a 96-well U-bottom plate (Becton Dickinson), and the reaction is performed on ice for 15 minutes. Thereafter, the reaction mixture to which 100 μl of effector cells have been added is incubated for 4 hours in a carbon dioxide incubator. The final concentration of the test antibody can be appropriately used within the range of 0 to 10 μg / ml. After the incubation, 100 μl of the supernatant is collected, and the radioactivity of the supernatant is measured with a gamma counter (COBRAII AUTO-GAMMA, MODEL D5005, Packard Instrument Company). Cytotoxic activity (%) can be calculated based on the formula (A−C) / (B−C) × 100 using the obtained radioactivity value. A is the radioactivity (cpm) when using a sample of each test antibody, B is the radioactivity (cpm) when using a sample with 1% NP-40 (nacalai tesque) added, and C is the target cell only The radioactivity (cpm) when a sample containing is used is shown.

  On the other hand, in the case of measuring CDC activity, 50 μl each of target cells and test antibody are added to a 96-well flat bottom plate (Becton Dickinson), and the reaction is carried out on ice for 15 minutes. Thereafter, the reaction mixture to which 100 μl of complement solution has been added is incubated for 4 hours in a carbon dioxide incubator. The final concentration of the test antibody can be appropriately used within the range of 0 to 3 μg / ml. After culture, 100 μl of the supernatant is collected, and the radioactivity of the supernatant is measured with a gamma counter. Cytotoxic activity can be calculated in the same way as measuring ADCC activity.

  On the other hand, when measuring cytotoxic activity using antibody conjugate, 50 μl each of target cell and test antibody conjugate is added to 96-well flat-bottom plate (Becton Dickinson), and reaction is carried out on ice for 15 minutes. Is done. The plate is incubated for 1 to 4 hours in a carbon dioxide incubator. The final concentration of the antibody can be appropriately used within the range of 0 to 3 μg / ml. After culture, 100 μl of the supernatant is collected, and the radioactivity of the supernatant is measured with a gamma counter. Cytotoxic activity can be calculated in the same way as measuring ADCC activity.

  The variable regions of the H chain and L chain of an antibody are usually composed of three CDRs and four FRs as described above. In a preferred embodiment of the present invention, the amino acid residue subjected to “modification” can be appropriately selected from, for example, amino acid residues constituting CDR or FR.

  Moreover, those skilled in the art suitably obtain the amino acid sequence that constitutes the FR of the variable region of an antibody and that actually exists in an organism such as human or mouse, using a public database such as Kabat. be able to.

  In a preferred embodiment of the present invention, a humanized antibody having controlled plasma pharmacokinetics by the method of the present invention is provided. The humanized antibody is, for example, a humanized antibody comprising a complementarity determining region (CDR) derived from a non-human animal, a framework region (FR) derived from human and a human C region, and the antibody surface in CDR or FR At least one amino acid residue that can be exposed to the amino acid is an amino acid residue having a different charge from the amino acid residue at the corresponding position of the CDR or FR of the original antibody, and in plasma compared to a chimeric antibody having the same C region It is a humanized antibody with controlled pharmacokinetics.

  Furthermore, in a preferred embodiment of the present invention, a human antibody having plasma pharmacokinetics controlled by the method of the present invention is provided. The human antibody is a human antibody comprising, for example, a human-derived complementarity determining region (CDR), a human-derived framework region (FR) and a human C region, and at least the human antibody can be exposed on the antibody surface in the CDR or FR. One amino acid residue is an amino acid residue having a different charge from the corresponding amino acid residue in the CDR or FR of the original antibody, and plasma pharmacokinetics are controlled compared to a chimeric antibody having the same C region. Human antibody.

  The human constant region preferably refers to a region containing a wild type human Fc region, but modified Fc can also be suitably used. The “altered Fc” may include those in which the amino acid residues constituting the Fc are altered, and may include those in which modifications made to the Fc portion are altered. A preferred example of the modification modification is to modify the sugar chain modification mode added to the Fc moiety. In this specification, “an antibody having a reduced content of fucose bound to the Fc region of an antibody” specifically disclosed as a reference example can be cited as such a preferred specific example.

  “An antibody having a reduced content of fucose bound to the antibody Fc region” refers to an antibody that has a significantly smaller amount of fucose bound and preferably cannot be detected when compared with a control antibody. Normally, fucose is added to N-glycoside-linked sugar chains that are bound to two sugar chain-binding sites present in the Fc region of the two H-chains constituting one antibody molecule. In the present invention, “an antibody having a reduced content of fucose bound to an antibody Fc region” refers to 50% or less, preferably 25% or less of the total sugar chain content of the control antibody when compared with such a normal antibody as a control. More preferably, it refers to an antibody having a fucose content of 10% or less, particularly preferably 0% or less. The fucose content can be measured by using an analysis method specifically shown as a reference example below. The method for producing an antibody having a reduced fucose content is described as a reference example of the present invention. For example, a method using an animal cell lacking fucosyltransferase (Biotechnol Bioeng. (2004), 87 (5), 614-22), a method of producing an animal cell with modified complex branched sugar chain modification (Biotechnol Bioeng. (2006) 93 (5), 851-61), and the like. In addition, as a method for preparing cells other than animal cells as host cells, a method using plant cells (Nature Biotechnology (2006) 24, 1591-7) or a method using yeast cells (Nature Biotechnology (2006) 24, 210-5) and the like can also be preferably mentioned.

A preferred embodiment of the production method of the present invention is a method for producing a polypeptide comprising an antibody variable region with a modified isoelectric point,
(A) modifying a nucleic acid encoding a polypeptide containing the amino acid residue so that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide is converted;
(B) culturing the host cell to express the nucleic acid;
(C) A method comprising recovering a polypeptide containing an antibody variable region from a host cell culture.
A preferred embodiment of the production method of the present invention is a method for producing a polypeptide comprising an antibody variable region with controlled plasma pharmacokinetics,
(A) modifying a nucleic acid encoding a polypeptide containing the amino acid residue so that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide is converted;
(B) culturing the host cell to express the nucleic acid;
(C) A method comprising recovering a polypeptide containing an antibody variable region from a host cell culture.
Furthermore, a polypeptide comprising a variable region of an antibody with controlled plasma pharmacokinetics produced by the method is also included in the present invention.

The present invention also provides a method for producing a multispecific polypeptide comprising a first polypeptide having a variable region of an antibody and a second polypeptide. The present invention further provides multispecific polypeptides produced by the method. In a preferred embodiment of the production method of the present invention, the nucleic acid encoding the amino acid residue of the first polypeptide and the second polypeptide so that the difference in isoelectric point between the first polypeptide and the second polypeptide is increased. A method comprising modifying both or any one of the nucleic acids encoding amino acid residues of a polypeptide. That is, by changing the charge of the amino acid residues of the first polypeptide and the second polypeptide, the difference in isoelectric point (pI) is increased in the polypeptide, and the difference between the isoelectric points is used for multiplexing. Specific antibodies can be produced. Specifically, the production method includes the following steps (a) to (c).
(A) a nucleic acid encoding a polypeptide containing the amino acid residue, so that the charge of at least one amino acid residue that can be exposed on the surface of the CDR regions of the first polypeptide and the second polypeptide is modified. The amino acid residues of the first polypeptide such that the modification of the nucleic acid increases the difference in isoelectric point between the first polypeptide and the second polypeptide compared to before modification. A nucleic acid encoding and / or a nucleic acid encoding an amino acid residue of a second polypeptide,
(B) culturing the host cell such that the nucleic acid is expressed;
(C) recovering the multispecific antibody from the host cell culture.

The polypeptide in the present invention usually refers to peptides and proteins having a length of about 10 amino acids or more. Moreover, although it is normally a polypeptide derived from a living organism | raw_food, it will not specifically limit, For example, the polypeptide which consists of a sequence designed artificially may be sufficient. Further, it may be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. Furthermore, fragments of the above polypeptides are also included in the polypeptides of the present invention.
In the present invention, the “multispecific polypeptide comprising a first polypeptide having a variable region of an antibody and a second polypeptide” means an antibody that binds to at least two different antigens or different epitopes within the same antigen. As described above, the polypeptide including the variable region of the antibody and including the variable region of the antibody includes, for example, an antibody, a low molecular weight antibody, a Scaffold protein, and the like.

  In the present invention, “the difference in isoelectric points of polypeptides increases” means that the isoelectric points of the two or more polypeptides are not equal to each other by modifying the surface amino acid charges, or This refers to increasing the isoelectric point difference between two or more polypeptides. The difference in isoelectric point can be observed by using a technique such as isoelectric focusing. In the present invention, it is preferable to control the isoelectric point while maintaining the structure and function (activity) of the polypeptide.

That is, the present invention provides a method for producing a multispecific polypeptide comprising a first polypeptide having a variable region of an antibody and a second polypeptide,
(A) at least one that can be exposed on the surface of the CDR region such that the difference in isoelectric point between the first polypeptide and the second polypeptide is 1.0 or more, preferably 1.2 or more, more preferably 1.5 or more Modifying the nucleic acid encoding the amino acid residue of the first polypeptide and / or the nucleic acid encoding the amino acid residue of the second polypeptide so that the charge of the amino acid residue is modified,
(B) culturing the host cell such that the nucleic acid is expressed;
(C) providing a method for producing a multispecific antibody comprising recovering the multispecific antibody from a host cell culture.

The present invention also provides a method for modifying a multispecific polypeptide for purifying a multispecific polypeptide comprising a first polypeptide having a variable region of an antibody and a second polypeptide. In a preferred embodiment of the purification method of the present invention, the nucleic acid encoding the amino acid residue of the first polypeptide and the second polypeptide so that the difference in isoelectric point between the first polypeptide and the second polypeptide is increased. A method comprising modifying both or any one of the nucleic acids encoding amino acid residues of a polypeptide. That is, by changing the charge of the amino acid residues of the first polypeptide and the second polypeptide, a difference in isoelectric point (pI) is introduced into the polypeptide, and the difference between the isoelectric points is used to multiplex the polypeptide. Specific antibodies can be purified. Specifically, the purification method includes the following steps (a) to (c).
(A) a nucleic acid encoding a polypeptide containing the amino acid residue, so that the charge of at least one amino acid residue that can be exposed on the surface of the CDR regions of the first polypeptide and the second polypeptide is modified. The amino acid residues of the first polypeptide such that the modification of the nucleic acid increases the difference in isoelectric point between the first polypeptide and the second polypeptide compared to before modification. A nucleic acid encoding and / or a nucleic acid encoding an amino acid residue of a second polypeptide,
(B) culturing the host cell such that the nucleic acid is expressed;
(C) purifying the multispecific antibody from the host cell culture by standard chromatography.

  In addition, the manufacturing method of the multispecific antibody including the process refine | purified with the said purification method is also contained in this invention.

  In the above method of the present invention, “modifying a nucleic acid” means modifying a nucleic acid sequence so as to be a codon corresponding to an amino acid residue introduced by “modification” in the present invention. More specifically, it means that the nucleic acid constituting the codon to be used for modification is modified so that the codon corresponding to the amino acid residue before modification becomes the codon of the amino acid residue introduced by modification. Usually, it means that genetic manipulation or mutation treatment is performed to replace at least one base of the nucleic acid constituting the codon so as to be a codon encoding the target amino acid residue. That is, a codon encoding an amino acid residue subjected to modification is replaced by a codon encoding an amino acid residue introduced by modification. Such nucleic acid modification can be appropriately performed by those skilled in the art using known techniques such as site-directed mutagenesis and PCR mutagenesis.

  The nucleic acid in the present invention is usually retained (inserted) in an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, if Escherichia coli is used as a host, a cloning vector such as pBluescript vector (Stratagene) is preferable, but various commercially available vectors can be used. The following vectors are available. Expression vectors are particularly useful when vectors are used to produce the polypeptides of the invention. The expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in E. coli, in cultured cells, or in an individual organism, for example, pBEST vector (Promega) for in vitro expression, PET vector (Invitrogen) for E. coli, pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, pME18S vector (Mol Cell Biol. (1988) 8, 466-472) for individual organisms, etc. preferable. The insertion of the DNA of the present invention into a vector can be suitably performed by a conventional method, for example, a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley). & Sons, Section 11.4-11.11).

  There is no restriction | limiting in particular as said host cell, According to the objective, various host cells are used. Examples of cells for expressing the polypeptide include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, Drosophila S2). Spodoptera SF9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells. Vector introduction into host cells includes, for example, calcium phosphate precipitation, electric pulse perforation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection, It can be performed by a known method such as an injection method.

  In order to secrete the polypeptide (antibody) expressed in the host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, an appropriate secretion signal can be suitably incorporated into the antibody of interest. As these signals, endogenous signals peculiar to the target polypeptide (antibody) or heterologous signals can be preferably used.

  Recovery of the polypeptide (antibody) in the above production method is carried out by recovering the medium when the polypeptide (antibody) of the present invention is secreted into the medium. When the antibody of the present invention is produced intracellularly, the cell is first lysed, and then the antibody is recovered.

  For purification of antibodies of the invention recovered from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography In addition, known methods including hydroxylapatite chromatography and lectin chromatography can be suitably used.

  In the present invention, the polypeptide that modifies the nucleic acid is preferably a homomultimer of the first polypeptide, a homomultimer of the second polypeptide, and a heteromultimer of the first polypeptide and the second polypeptide. Is the body. Examples of the homomultimer of the first polypeptide, the homomultimer of the second polypeptide, and the heteromultimer of the first polypeptide and the second polypeptide include those described in Examples. It is not limited to.

  Standard chromatography in the present invention includes, but is not limited to, cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, hydrophobic charge interaction chromatography, and chromatofocusing. Not.

  In the above method of the present invention, the first polypeptide and the second polypeptide preferably include a heavy chain variable region (VH). The variable region may include, for example, a complementarity determining region (CDR) and a framework region (FR).

  Furthermore, in the above method of the present invention, the variable region of the multispecific polypeptide preferably includes a light chain variable region.

Furthermore, in the above method of the present invention, the first polypeptide and the second polypeptide preferably include a heavy chain constant region. The heavy chain constant region is more preferably one in which a pI difference occurs between the first polypeptide and the second polypeptide. Such a heavy chain constant region includes the heavy chain constant region of an antibody having a pI difference, and the first and second heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4 that originally differ in pI are used. PI differences can be introduced into two polypeptides, only amino acids resulting from isoelectric point differences between these subclasses in the heavy chain constant regions in the first and second polypeptides, or the like A non-wild type human constant region can be prepared by simultaneously modifying adjacent amino acids that do not affect the electric point, and a pI difference can be introduced between the two constant regions. Examples of modifications for introducing a pI difference into the constant region include EU numbering of the H chain constant region, H chain 137th, 196th, 203rd, 214th, 217th, 233rd, 268th, 274th 276, 297, 355, 392, 419, and 435.
In addition, since the pI difference is generated by removing the sugar chain in the heavy chain constant region, the 297th position of the sugar chain addition site is also exemplified as a modified site for introducing the pI chain.
In addition, in the present invention, in contrast to the method in which the first polypeptide and the second polypeptide include a heavy chain constant region, the first polypeptide and the second polypeptide include a heavy chain variable region. Method and / or wherein the multispecific antibody comprises a third polypeptide comprising a light chain variable region, wherein the first polypeptide and the second polypeptide each comprise the third polypeptide and a multimer. A method in combination with a forming method is also included.

  Furthermore, multispecific polypeptides produced by the above method are also included in the present invention.

  Further, when the first polypeptide in the multispecific antibody provided by the present invention contains a heavy chain variable region, for example, in order to make the “isoelectric point difference increase”, for example, Examples include an embodiment in which at least one amino acid residue selected from the amino acid residues at positions 31, 61, 62, 64, and 65 by Kabat numbering in the chain variable region is charged. Further, when the light chain variable region is included, in order to make the above “so that the difference in isoelectric point is increased”, for example, positions 24, 27, 53 by Kabat numbering in the light chain variable region, Examples include an embodiment in which at least one amino acid residue selected from the 54th and 55th amino acid residues is charged. Among the amino acid residues of the first polypeptide indicated by the above numbering, amino acid residues other than the amino acid residue having the charge are different in isoelectric point between the first polypeptide and the second polypeptide. If it is, the same kind of charge as the amino acid residue having the charge may be used, or it may be the opposite charge or not.

  The multispecific antibody of the present invention is preferably characterized in that the second polypeptide has a charge opposite to the charged amino acid residue of the first polypeptide or no charge. . Specifically, the second polypeptide includes a heavy chain variable region, and is selected from amino acid residues at positions 31, 61, 62, 64, and 65 by Kabat numbering in the heavy chain variable region. A multispecific antibody wherein the amino acid residue is selected in the variable region contained in the first polypeptide, has a charge opposite to the amino acid residue to be charged, or has no charge. When the light chain variable region is included, at least one amino acid residue selected from the amino acid residues at positions 24, 27, 53, 54 and 55 by Kabat numbering in the light chain variable region is A multispecific antibody that is selected in the variable region contained in the first polypeptide, has a charge opposite to the amino acid residue to be charged, or has no charge. Among the amino acid residues of the second polypeptide indicated by the above numbering, amino acid residues other than the charged amino acid residue are different in isoelectric point between the first polypeptide and the second polypeptide. If it is, the same kind of charge as the amino acid residue having the charge may be used, or it may be the opposite charge or not.

Further, when the multispecific antibody contains an antibody constant region, in order to reduce its isoelectric point, for example, position 137 is an IgG2 or IgG4 sequence, position 196 is an IgG1 or IgG2 or IgG4 sequence, position 203 Is an IgG2 or IgG4 sequence, 214 is an IgG2 sequence, 217 is an IgG1 or IgG3 or IgG4 sequence, 233 is an IgG1 or IgG3 or IgG4 sequence, 268 is an IgG4 sequence, 274 is an IgG2 or IgG3 or IgG4 sequence, position 276 is IgG1 or IgG2 or IgG4 sequence, position 355 is IgG4 sequence, position 392 is IgG3 sequence, position 419 is IgG4 sequence, position 435 is IgG1 or IgG2 or IgG4 sequence It is desirable. In order to increase the isoelectric point, for example, position 137 is an IgG1 or IgG3 sequence, position 196 is an IgG3 sequence, position 203 is an IgG1 or IgG3 sequence, position 214 is an IgG1 or IgG3 or IgG4 sequence, 217 is an IgG2 sequence, 233 is an IgG2 sequence, 268 is an IgG1 or IgG2 or IgG3 sequence, 274 is an IgG1 sequence, 276 is an IgG3 sequence, 355 is an IgG1 or IgG2 or IgG3 sequence, It is desirable to apply an IgG1 or IgG2 or IgG4 sequence at position 392, an IgG1 or IgG2 or IgG3 sequence at position 419, and an IgG3 sequence at position 435.
Application of these sequences is not limited as long as a sufficient isoelectric point difference is given to both H chains, and not all sequences need to be applied.

In the above antibody, “having the same kind of charge” means, for example, any of the amino acid residues by the Kabat numbering in the heavy chain variable region or the amino acid residues by the EU numbering in the heavy chain constant region as described below (a ) Or (b) means having an amino acid residue included in any one group.
(A) Glutamic acid (E), aspartic acid (D)
(B) Lysine (K), Arginine (R), Histidine (H)

  Further, “having an opposite charge” means, for example, at least one of the amino acid residues by the Kabat numbering or the EU numbering in the second polypeptide having the heavy chain variable region and / or the heavy chain constant region. The amino acid residue is an amino acid residue at a corresponding position in the heavy chain variable region and / or heavy chain constant region contained in the first polypeptide, and the group of any one of (a) or (b) above Means that the remaining amino acid residues have amino acid residues included in different groups.

  That is, the present invention provides a multispecific antibody wherein the amino acid residues having the same type of charge are selected from the amino acid residues included in any one of the groups (a) and (b).

  In addition, when the original amino acid residue (before modification) has already been charged, it is also a preferred embodiment of the present invention to modify the amino acid residue to have no charge.

  In the present invention, the amino acid residue is preferably modified so that the difference in isoelectric point (pI) between the first polypeptide and the second polypeptide increases. In addition, when there are a plurality of amino acid residues introduced by modification, these amino acid residues may contain a small number of amino acid residues having no charge.

  The present invention also relates to a composition (drug) comprising a polypeptide (for example, an antibody such as an IgG antibody) whose plasma pharmacokinetics is controlled by the method of the present invention, and a pharmaceutically acceptable carrier.

  In the present invention, the pharmaceutical composition usually refers to a drug for treatment or prevention of a disease, or examination / diagnosis.

  The pharmaceutical composition of the present invention can be suitably formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or as an injection in suspension. For example, a pharmacologically acceptable carrier or medium, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative , Suitably combined with binders and the like, and can be suitably formulated by mixing in unit dosage forms generally required for accepted pharmaceutical practice. The amount of the active ingredient in these preparations is set so that an appropriate dose within the indicated range can be obtained.

  Sterile compositions for injection can be suitably formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

  Examples of the aqueous solution for injection include isotonic solutions containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride). A suitable solubilizer such as alcohol (ethanol, etc.), polyalcohol (propylene glycol, polyethylene glycol, etc.), nonionic surfactant (polysorbate 80 (TM), HCO-50, etc.) can be suitably used in combination.

  Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol can be suitably used as a solubilizer. Moreover, a buffer (for example, phosphate buffer and sodium acetate buffer), a soothing agent (for example, procaine hydrochloride), a stabilizer (for example, benzyl alcohol and phenol), and an antioxidant can be suitably blended. The injection solution prepared as described above is usually filled in a suitable ampoule.

  The pharmaceutical composition of the present invention can be administered preferably by parenteral administration. For example, it can be suitably prepared as a composition for injection, nasal administration, pulmonary administration, and transdermal administration. For example, it can be appropriately administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

  The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be appropriately set within the range of 0.0001 mg to 1000 mg per kg body weight, for example. Alternatively, for example, the dose may be set or adjusted to 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these values. The dose and administration method vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can set an appropriate dose and administration method in consideration of these conditions.

  The present invention also provides a nucleic acid encoding an antibody (eg, humanized anti-glypican 3 antibody) whose plasma pharmacokinetics is controlled by the method of the present invention. Furthermore, a vector carrying the nucleic acid is also included in the present invention.

  Furthermore, the present invention provides a host cell containing the nucleic acid. The type of the host cell is not particularly limited, and examples thereof include bacterial cells such as E. coli and various animal cells. The host cell can be suitably used as a production system for production and expression of the antibody of the present invention. That is, the present invention provides a production system used for the production of an antibody using the host cell. As the production system, in vitro and in vivo production systems can be suitably used. As the host cell used in the in vitro production system, eukaryotic cells and prokaryotic cells are preferably used.

  Examples of eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. Animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108, 945), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero and the like amphibian cells such as Xenopus oocytes (Valle et al., Nature (1981) 291, 338-340) and insect cells such as Sf9, Sf21, and Tn5 are preferably exemplified. For the expression of the antibody of the present invention, CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells, and BHK cells are preferably used. CHO cells are particularly preferred as host cells for the purpose of mass expression by animal cells. For example, by using methods such as the calcium phosphate method, the DEAE dextran method, the method using the cationic ribosome DOTAP (Boehringer Mannheim), the electroporation method, the lipofection, etc., the introduction of the recombinant vector into the host cell is suitably carried out. Is done.

  As plant cells, for example, cells derived from Nicotiana tabacum and Lemna minor are known as protein production systems, and the antibody of the present invention can be produced by a method of callus culture of these cells. Fungal cells include yeast, for example, cells of the genus Saccharomyces (Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc.), and fungi, for example, cells of the genus Aspergillus A protein expression system using niger (Aspergillus niger, etc.) is known and can be used as a host cell for producing the antibody of the present invention.

  When prokaryotic cells are used, production systems using bacterial cells are preferably used. As a bacterial cell, a production system using B. subtilis in addition to the aforementioned E. coli is known, and any of these bacterial cells is suitable for producing the antibody of the present invention. Can be used.

  In order to produce an antibody using the host cell of the present invention, a host cell transformed with an expression vector containing a polynucleotide encoding the antibody of the present invention is cultured, and the polynucleotide encoding the antibody in the culture Is expressed. The culture can be suitably performed according to a known method. For example, when animal cells are used as the host, for example, DMEM, MEM, RPMI1640, and IMDM can be suitably used as the culture solution. In that case, serum supplements such as FBS and fetal calf serum (FCS) are preferably used in combination. In addition, cells can be cultured by serum-free culture. Although it depends on the host cell, it can be suitably cultured under conditions of pH of about 6 to 8 at the time of culture. The culture is usually performed at about 30 to 40 ° C. for about 15 to 200 hours, and medium exchange, aeration, and agitation are added as necessary.

  On the other hand, as a system for producing the antibody of the present invention in vivo, for example, a production system using animals or a production system using plants can be preferably used. That is, the polynucleotide encoding the antibody of the present invention is introduced into these animals or plants, and the anti-glypican 3 antibody is produced and collected in the body of the animals or plants. The “host” in the present invention includes these animals and plants.

  When animals are used as hosts, production systems using mammals and insects can be used. As mammals, goats, pigs, sheep, mice, cows and the like are preferably used (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). Moreover, when a mammal is used, a transgenic animal is used.

  For example, a polynucleotide encoding the antibody of the present invention is prepared as a fusion gene with a gene encoding a polypeptide specifically produced in milk, such as goat β casein. Next, a polynucleotide fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. The antibody of interest is obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its offspring. In order to increase the amount of milk containing the antibody produced from the transgenic goat, a hormone is suitably administered to the transgenic goat as appropriate (Ebert et al., Bio / Technology (1994) 12, 699-702). .

  Moreover, as an insect which produces the antibody of this invention, a silkworm can be used, for example. When a silkworm is used, a baculovirus in which a polynucleotide encoding the antibody of interest is inserted into the viral genome is used in the infection against the silkworm. The desired anti-glypican 3 antibody is obtained from the body fluid of the infected silkworm (Susumu et al., Nature (1985) 315, 592-4).

  Furthermore, when a plant is used for production of the antibody of the present invention, for example, tobacco can be used as the plant. When tobacco is used, a recombinant vector obtained as a result of inserting a polynucleotide encoding the antibody of interest into a plant expression vector, such as pMON 530, is used in bacteria such as Agrobacterium tumefaciens. Can be introduced. The bacterium is used for infection against tobacco, for example Nicotiana tabacum (Ma et al., Eur. J. Immunol. (1994) 24, 131-8) and desired from the leaves of infected tobacco. Glypican 3 antibody is obtained. In addition, similar bacteria are used for infection of duckweed (Lemna minor), and the desired anti-glypican 3 antibody can be obtained from cloned infected duckweed cells (Cox KM et al. Nat. Biotechnol. (2006) 24 (12). ), 1591-7).

  The antibody of the present invention thus obtained can be isolated from inside or outside the host cell (medium, milk, etc.) and purified as a substantially pure and homogeneous antibody. For the separation and purification of the antibody, the separation and purification methods used in usual polypeptide purification can be suitably used, but are not limited thereto. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected, In combination, the antibodies can be suitably separated and purified.

  Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshaket al. (1996) Cold Spring Harbor Laboratory Press). These chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. Examples of the column using protein A include Hyper D, POROS, Sepharose F. F. (Pharmacia) and the like.

  A method for producing the antibody of the present invention with controlled plasma pharmacokinetics comprising the steps of culturing the host cell of the present invention as described above and recovering glypican 3 antibody from the culture of the cell is also provided by the present invention. This is one of the preferred embodiments.

  The present invention modifies the amino acid sequences of the variable and constant regions of TOCILIZUMAB, which is a humanized anti-IL-6 receptor IgG1 antibody, to enhance the drug retention and improve the plasma retention while enhancing the drug retention. A pharmaceutical composition comprising a second generation molecule that exhibits a therapeutic effect continuously and improves immunogenicity, safety, and physical properties, and is superior to TOCILIZUMAB, and a method for producing these pharmaceutical compositions. provide. Furthermore, the present invention provides antibody constant regions suitable for use as pharmaceuticals.

The present invention relates to an anti-IL-6 receptor antibody having excellent antigen binding activity, neutralizing activity, plasma retention, stability and / or homogeneity, and reducing the risk of immunogenicity.
Preferably, the anti-IL-6 receptor antibody is a humanized PM-1 antibody (TOCILIZUMAB). More specifically, the present invention relates to a humanized PM-1 antibody having enhanced antigen binding activity by amino acid substitution, a humanized PM-1 antibody having enhanced neutralizing activity, and humanized PM-1 having improved plasma retention. Antibodies, humanized PM-1 antibodies with reduced risk of immunogenicity, humanized PM-1 antibodies with improved stability, and humanized PM-1 antibodies with improved homogeneity are provided.

Humanized PM-1 antibody binds to the human IL-6 receptor and inhibits the binding of human IL-6 and human IL-6 receptor. In the present specification, the correspondence between the amino acid sequence of the humanized PM-1 antibody and the SEQ ID NO of the sequence listing is as follows.
Heavy chain amino acid sequence SEQ ID NO: 15
Light chain amino acid sequence SEQ ID NO: 16
Amino acid sequence of heavy chain variable region SEQ ID NO: 17
Amino acid sequence of light chain variable region SEQ ID NO: 18
Amino acid sequence of heavy chain CDR1 (HCDR1) SEQ ID NO: 1
Amino acid sequence of heavy chain CDR2 (HCDR2) SEQ ID NO: 2
Amino acid sequence of heavy chain CDR3 (HCDR3) SEQ ID NO: 3
Amino acid sequence of heavy chain FR1 (HFR1) SEQ ID NO: 7
Amino acid sequence of heavy chain FR2 (HFR2) SEQ ID NO: 8
Amino acid sequence of heavy chain FR3 (HFR3) SEQ ID NO: 9
Amino acid sequence of heavy chain FR4 (HFR4) SEQ ID NO: 10
The amino acid sequence of the light chain CDR1 (LCDR1) is SEQ ID NO: 4.
The amino acid sequence of the light chain CDR2 (LCDR2) is SEQ ID NO: 5.
The amino acid sequence of the light chain CDR3 (LCDR3) is shown in SEQ ID NO: 6.
The amino acid sequence of the light chain FR1 (LFR1) is shown in SEQ ID NO: 11
The amino acid sequence of the light chain FR2 (LFR2) is shown in SEQ ID NO: 12
The amino acid sequence of the light chain FR3 (LFR3) is shown in SEQ ID NO: 13
The amino acid sequence of the light chain FR4 (LFR4) is shown in SEQ ID NO: 14

<Affinity / neutralizing activity enhancing antibody>
The present invention provides an anti-human IL-6 receptor antibody having high binding activity and / or neutralizing activity for human IL-6 receptor. More specifically, the present invention provides the antibodies described in (a) to (y) below and a method for producing the antibodies.
(a) An anti-human IL-6 receptor antibody having a heavy chain CDR1 in which the first Ser is substituted with another amino acid in the amino acid sequence (HCDR1) described in SEQ ID NO: 1.
The amino acid after substitution is not particularly limited, but Trp (RD_68), Thr (RD_37), Asp (RD_8), Asn (RD_11), Arg (RD_31), Val (RD_32), Phe (RD_33), Ala (RD_34), Substitution with Gln (RD_35), Tyr (RD_36), Leu (RD_38), His (RD_42), Glu (RD_45) or Cys (RD_46) is preferred.
The sequence in which the first Ser is replaced with Trp in the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 26.
The sequence in which the first Ser is replaced with Thr in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 27.
The sequence in which the first Ser is replaced with Asp in the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 28.
The sequence in which the first Ser is substituted with Asn in the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 29.
A sequence in which the first Ser is substituted with Arg in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 30.
The sequence in which the first Ser is replaced with Val in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 31.
The sequence in which the first Ser is replaced with Phe in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 32.
The sequence in which the first Ser is replaced with Ala in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 33.
The sequence in which the first Ser is replaced with Gln in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 34.
The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Tyr is shown in SEQ ID NO: 35.
The sequence in which the first Ser is replaced with Leu in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 36.
A sequence in which the first Ser is replaced with His in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 37.
The sequence in which the first Ser is substituted with Glu in the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 38.
A sequence in which the first Ser is substituted with Cys in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 39.

(b) An anti-human IL-6 receptor antibody having a heavy chain CDR1 in which the fifth Trp is substituted with another amino acid in the amino acid sequence (HCDR1) described in SEQ ID NO: 1.
The amino acid after substitution is not particularly limited, but substitution to Ile (RD_9) or Val (RD_30) is preferable.
The sequence in which the 5th Trp is replaced with Ile in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 40.
The sequence in which the 5th Trp is substituted with Val in the amino acid sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 41.

(c) An anti-human IL-6 receptor antibody having a heavy chain CDR2 in which the first Tyr is substituted with another amino acid in the amino acid sequence (HCDR2) described in SEQ ID NO: 2.
The amino acid after substitution is not particularly limited, but substitution to Phe (RD_82) is preferred.
The sequence in which the first Tyr is substituted with Phe in the amino acid sequence described in SEQ ID NO: 2 is shown in SEQ ID NO: 42.

(d) An anti-human IL-6 receptor antibody having a heavy chain CDR2 in which the 8th Thr is substituted with another amino acid in the amino acid sequence (HCDR2) described in SEQ ID NO: 2.
The amino acid after substitution is not particularly limited, but substitution to Arg (RD_79) is preferred.
A sequence in which the 8th Thr is substituted with Arg in the amino acid sequence described in SEQ ID NO: 2 is shown in SEQ ID NO: 43.

(e) An anti-human IL-6 receptor antibody having a heavy chain CDR2 in which the 9th Thr is substituted with another amino acid in the amino acid sequence (HCDR2) described in SEQ ID NO: 2.
The amino acid after substitution is not particularly limited, but substitution to Ser (RD_12) or Asn (RD_61) is preferred.
The sequence in which the 9th Thr is substituted with Ser in the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 44.
The sequence in which the 9th Thr is substituted with Asn in the amino acid sequence described in SEQ ID NO: 2 is shown in SEQ ID NO: 45.

(f) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the first Ser is substituted with another amino acid in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3.
The amino acid after substitution is not particularly limited, but substitution to Ile (RD_2), Val (RD_4), Thr (RD_80) or Leu (RD_5) is preferable.
In the amino acid sequence shown in SEQ ID NO: 3, the sequence in which the first Ser is substituted with Ile is shown in SEQ ID NO: 46.
The sequence in which the first Ser is replaced with Val in the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 47.
The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 3 is replaced with Thr is shown in SEQ ID NO: 48.
The sequence in which the first Ser is replaced with Leu in the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 49.

(g) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the second Leu is substituted with another amino acid in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3.
The amino acid after substitution is not particularly limited, but substitution to Thr (RD_84) is preferred.
The sequence in which the second Leu in the amino acid sequence described in SEQ ID NO: 3 is replaced with Thr is shown in SEQ ID NO: 50.

(h) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the fifth Thr is substituted with another amino acid in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3.
The amino acid after substitution is not particularly limited, but substitution to Ala (RD_3) or Ile (RD_83) is preferable. Another preferred substitution includes substitution of the fifth Thr with Ser (RDC — 14H).
The sequence in which the fifth Thr is substituted with Ala in the amino acid sequence set forth in SEQ ID NO: 3 is shown in SEQ ID NO: 51.
The sequence in which the fifth Thr is substituted with Ile in the amino acid sequence set forth in SEQ ID NO: 3 is shown in SEQ ID NO: 52.
In the amino acid sequence shown in SEQ ID NO: 3, the sequence in which the 5th Thr is substituted with Ser is shown in SEQ ID NO: 53.

(i) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the 7th Ala is substituted with another amino acid in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3.
The amino acid sequence after substitution is not particularly limited, but substitution to Ser (RD_81) or Val (PF_3H) is preferable.
In the amino acid sequence shown in SEQ ID NO: 3, the sequence in which the 7th Ala is substituted with Ser is shown in SEQ ID NO: 54.
The sequence in which the 7th Ala is substituted with Val in the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 55.

(j) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the 8th Met is substituted with another amino acid in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3.
The amino acid sequence after substitution is not particularly limited, but substitution with Leu (PF_4H) is preferred.
The sequence in which the 8th Met in the amino acid sequence described in SEQ ID NO: 3 is replaced with Leu is shown in SEQ ID NO: 56.

(k) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the first Ser and the fifth Thr are substituted with other amino acids in the amino acid sequence (HCDR3) described in SEQ ID NO: 3.
The amino acid after substitution is not particularly limited, but it is preferable to substitute the first Ser with Leu and the fifth Thr with Ala (RD_6). As other preferable substitutions, the substitution of the first Ser to Val and the substitution of the fifth Thr to Ala (RDC_2H), the substitution of the first Ser to Ile and the substitution of Ala to the fifth Thr (RDC_3H), replacement of the first Ser with Thr and replacement of the fifth Thr with Ala (RDC_4H), replacement of the first Ser with Val and replacement of the fifth Thr with Ile (RDC_5H), 1st Ser to Ile and 5th Thr to Ile (RDC_6H), 1st Ser to Thr and 5th Thr to Ile (RDC_7H), or 1st Substitution of Ser to Leu and substitution of 5th Thr to Ile (RDC_8H) can be mentioned.
A sequence in which the first Ser is replaced with Leu and the fifth Thr is replaced with Ala in the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 57.
In the amino acid sequence shown in SEQ ID NO: 3, the sequence in which the first Ser is replaced with Val and the fifth Thr is replaced with Ala is shown in SEQ ID NO: 58.
In the amino acid sequence shown in SEQ ID NO: 3, the sequence in which the first Ser is replaced with Ile and the fifth Thr is replaced with Ala is shown in SEQ ID NO: 59.
In the amino acid sequence shown in SEQ ID NO: 3, the sequence in which the first Ser is replaced with Thr and the fifth Thr is replaced with Ala is shown in SEQ ID NO: 60.
A sequence in which the first Ser is replaced with Val and the fifth Thr is replaced with Ile in the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 61.
In the amino acid sequence shown in SEQ ID NO: 3, the sequence in which the first Ser is replaced with Ile and the fifth Thr is replaced with Ile is shown in SEQ ID NO: 62.
In the amino acid sequence shown in SEQ ID NO: 3, the sequence in which the first Ser is replaced with Thr and the fifth Thr is replaced with Ile is shown in SEQ ID NO: 63.
In the amino acid sequence shown in SEQ ID NO: 3, the sequence in which the first Ser is replaced with Leu and the fifth Thr is replaced with Ile is shown in SEQ ID NO: 64.

(l) an anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the second Leu, the seventh Ala and the eighth Met are substituted with other amino acids in the amino acid sequence (HCDR3) described in SEQ ID NO: 3 .
The amino acid after substitution is not particularly limited, but it is preferable to substitute the second Leu with Thr, the seventh Ala with Val, and the eighth Met with Leu (RD_78).
The sequence in which the second Leu is replaced with Thr, the seventh Ala with Val, and the eighth Met with Leu in the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 65.

(m) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the first Arg is substituted with another amino acid in the amino acid sequence (LCDR1) described in SEQ ID NO: 4.
The amino acid sequence after substitution is not particularly limited, but substitution to Phe (RD_18) is preferred.
The sequence in which the first Arg is substituted with Phe in the amino acid sequence set forth in SEQ ID NO: 4 is shown in SEQ ID NO: 66.

(n) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the fourth Gln is substituted with another amino acid in the amino acid sequence (LCDR1) described in SEQ ID NO: 4.
The amino acid sequence after substitution is not particularly limited, but substitution to Arg (RD_26) or Thr (RD_20) is preferred.
The sequence in which the 4th Gln is substituted with Arg in the amino acid sequence set forth in SEQ ID NO: 4 is shown in SEQ ID NO: 67.
The sequence in which the fourth Gln is substituted with Thr in the amino acid sequence described in SEQ ID NO: 4 is shown in SEQ ID NO: 68.

(o) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the 9th Tyr is substituted with another amino acid in the amino acid sequence (LCDR1) described in SEQ ID NO: 4.
The amino acid after substitution is not particularly limited, but substitution to Phe (RD_73) is preferred.
The sequence in which the 9th Tyr is substituted with Phe in the amino acid sequence set forth in SEQ ID NO: 4 is shown in SEQ ID NO: 69.

(p) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the 11th Asn is substituted with another amino acid in the amino acid sequence (LCDR1) described in SEQ ID NO: 4.
The amino acid sequence after substitution is not particularly limited, but substitution with Ser (RD_27) is preferred.
The sequence in which the 11th Asn is substituted with Ser in the amino acid sequence shown in SEQ ID NO: 4 is shown in SEQ ID NO: 70.

(q) An anti-human IL-6 receptor antibody having a light chain CDR2 in which the second Thr is substituted with another amino acid in the amino acid sequence (LCDR2) set forth in SEQ ID NO: 5.
The amino acid sequence after substitution is not particularly limited, but substitution with Gly is preferred.
The sequence in which the second Thr is replaced with Gly in the amino acid sequence set forth in SEQ ID NO: 5 is shown in SEQ ID NO: 71.

(r) An anti-human IL-6 receptor antibody having a light chain CDR3 in which the first Gln is substituted with another amino acid in the amino acid sequence (LCDR3) described in SEQ ID NO: 6.
The amino acid sequence after substitution is not particularly limited, but substitution to Gly (RD_28), Asn (RD_29) or Ser (RDC_15L) is preferable.
A sequence in which the first Gln is substituted with Gly in the amino acid sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 72.
The sequence in which the first Gln is substituted with Asn in the amino acid sequence set forth in SEQ ID NO: 6 is shown in SEQ ID NO: 73.
The sequence in which the first Gln is substituted with Ser in the amino acid sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 74.

(s) An anti-human IL-6 receptor antibody having a light chain CDR3 in which the third Gly is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 6.
The amino acid sequence after substitution is not particularly limited, but substitution with Ser is preferable.
In the amino acid sequence shown in SEQ ID NO: 6, the sequence in which the third Gly is replaced with Ser is shown in SEQ ID NO: 75.

(t) The light chain CDR1 in which the 9th Tyr is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 4 (LCDR1), and the 3rd Gly in the amino acid sequence described in SEQ ID NO: 6 (LCDR3) An anti-human IL-6 receptor antibody having a light chain CDR3 substituted with other amino acids.
The amino acid after substitution is not particularly limited, but the 9th Tyr in the amino acid sequence (LCDR1) described in SEQ ID NO: 4 is preferably substituted with Phe, and in the amino acid sequence (LCDR3) described in SEQ ID NO: 6 The third Gly is preferably replaced with Ser (RD_72).

(u) An anti-human IL-6 receptor antibody having a light chain CDR3 in which the fifth Thr is substituted with another amino acid in the amino acid sequence (LCDR3) set forth in SEQ ID NO: 6.
The amino acid after substitution is not particularly limited, but substitution to Arg (RD_23) or Ser is preferable.
The sequence in which the fifth Thr is substituted with Arg in the amino acid sequence set forth in SEQ ID NO: 6 is shown in SEQ ID NO: 76.
A sequence in which the 5th Thr is replaced with Ser in the amino acid sequence set forth in SEQ ID NO: 6 is shown in SEQ ID NO: 77.

(v) An anti-IL-6 receptor antibody having a light chain CDR3 in which the first Gln and the fifth Thr are substituted with other amino acids in the amino acid sequence (LCDR3) described in SEQ ID NO: 6.
The amino acid after substitution is not particularly limited, but it is preferable to substitute the first Gln with Gly and the fifth Thr with Ser (RD_22). Other preferred substitutions include the first substitution of Gln with Gly and the fifth substitution of Thr with Arg (RDC_11L).
In the amino acid sequence shown in SEQ ID NO: 6, the sequence in which the first Gln is replaced with Gly and the fifth Thr is replaced with Ser is shown in SEQ ID NO: 78.
In the amino acid sequence shown in SEQ ID NO: 6, the sequence in which the first Gln is replaced with Gly and the fifth Thr is replaced with Arg is shown in SEQ ID NO: 79.

(w) a heavy chain CDR2 in which the 9th Thr is substituted with another amino acid in the amino acid sequence (HCDR2) described in SEQ ID NO: 2, the first Ser in the amino acid sequence (HCDR3) described in SEQ ID NO: 3 An anti-IL-6 receptor antibody comprising a heavy chain CDR3 in which the fifth Thr is substituted with another amino acid.
The 9th Thr in the amino acid sequence (HCDR2) described in SEQ ID NO: 2 is preferably substituted with Asn. As preferred combinations of amino acids after substitution of the first Ser and the fifth Thr in the amino acid sequence (HCDR3) described in SEQ ID NO: 3, Leu and Ala (RDC_27H), Val and Ala (RDC_28H), Ile and Ala (RDC_30H), Thr and Ala (RDC_4H), Val and Ile (RDC_29H), Ile and Ile (RDC_32H), Thr and Ile (RDC_7H), Leu and Ile (RDC_8H).

(x) An antibody comprising a variable region having the heavy chain CDR3 according to (k) and a variable region having the light chain CDR3 according to (v).
(y) The antibody according to (x), further comprising the heavy chain CDR2 according to (e).

  The present invention provides an antibody comprising at least the amino acid substitution described in any of (a) to (y) above and a method for producing the antibody. Therefore, the antibody of the present invention includes antibodies containing amino acid substitutions other than the amino acid substitutions described in the above (a) to (y) in addition to the amino acid substitutions described in any of the above (a) to (y). included. The antibody of the present invention also includes an antibody in which a plurality of amino acid substitutions described in any of the above (a) to (y) are combined. Examples of the amino acid substitution described in the above (a) to (y) include substitution of the above CDR amino acid sequence with another amino acid. Examples of amino acid substitutions other than the amino acid substitutions described in (a) to (y) above include substitution, deletion, addition and / or insertion of the amino acid sequence of other CDR portions. Moreover, substitution, deletion, addition and / or insertion of the amino acid sequence of FR can be mentioned. Moreover, substitution, deletion, addition and / or insertion of the amino acid sequence of the constant region can be mentioned.

  The antibody of the present invention also includes an antibody obtained by transplanting the high affinity CDR found in the present invention into any framework other than the humanized PM-1 antibody. In addition, the antibody of the present invention includes an antibody having a reduced affinity as a result of transplantation of the high affinity CDR found in the present invention to a framework other than the humanized PM-1 antibody. Mutation is introduced into the CDR portion in order to obtain an antibody having a mutation introduced into the work portion (see, for example, Curr Opin Biotechnol. 1994 Aug; 5 (4): 428-33) and an antibody with the original affinity (eg, US2006 / 0122377).

  In the present invention, the amino acid substitution described in any of (a) to (y) above is preferably performed on the humanized PM-1 antibody. In the humanized PM-1 antibody, the antibody in which the amino acid substitution described in any of the above (a) to (y) is performed has high neutralizing activity against the IL-6 receptor. In the humanized PM-1 antibody, the antibody in which the amino acid substitution described in any of the above (a) to (y) is performed is used as a therapeutic agent for inflammatory diseases such as rheumatoid arthritis associated with IL-6. It is valid.

In addition, the antibody containing the amino acid substitution described in any of the above (a) to (y) can also be expressed as, for example, (1) or (2) below. Here, the antibody (a) is described as an example, but the antibodies (b) to (y) can also be expressed in the same manner.
(1) An antibody comprising a heavy chain variable region having CDR1 having an amino acid sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with another amino acid (2) as CDR1, SEQ ID NO: 1 An antibody comprising an H chain having an amino acid sequence in which the first Ser is substituted with another amino acid in the amino acid sequence described in 1.

<Antibodies with enhanced binding activity>
The present invention further provides an anti-IL-6 receptor antibody having a high binding activity to IL-6 receptor. In the present invention, the anti-IL-6 receptor antibody having a high binding activity to the IL-6 receptor is an antibody having an affinity of 1 nM or less, preferably 0.1 nM or less, usually at 37 ° C. under physiological conditions. More preferred is an antibody having an affinity of 0.04 nM or less. Such an anti-IL-6 receptor antibody having a high binding activity to the IL-6 receptor is considered to have improved neutralizing ability of the biological action of the antigen.
In the anti-IL-6 receptor antibody having a high binding activity to the IL-6 receptor of the present invention, the amino acid substitution introduced is not particularly limited, and examples thereof include the amino acid substitution described above.
The IL-6 receptor is not particularly limited, but human IL-6 receptor is preferred.
The binding activity can be measured by methods known to those skilled in the art. For example, it can be measured by Biacore (BIACORE) using SPR.

<Antibodies that reduce the immunogenicity risk of CDR sequences>
The present invention also provides anti-IL-6 receptor antibodies with reduced immunogenicity, particularly humanized PM-1 antibodies. It is believed that the presence of a T-cell epitope that binds to HLA in the antibody sequence increases the immunogenicity of the antibody. Therefore, the risk of immunogenicity of an antibody can be reduced by substituting the antibody sequence and removing the T-cell epitope present in the antibody sequence.

The present invention is a humanized anti-human IL-6 receptor antibody, particularly humanized, in which the T-cell epitope is removed by substituting the amino acid sequence of the antibody, particularly the CDR sequence with another amino acid, and the T-cell epitope is reduced. PM-1 light chain variable region is provided. The present invention also provides an antibody comprising the light chain variable region.
More specifically, the present invention provides a light chain CDR2 in which the second Thr is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 5 (LCDR2). The present invention also provides a light chain variable region comprising the light chain CDR2. The present invention also provides an anti-IL-6 receptor antibody comprising the light chain variable region. The amino acid sequence after substitution is not particularly limited, but substitution with Gly is preferred. The sequence in which the second Thr is replaced with Gly in the amino acid sequence set forth in SEQ ID NO: 5 is shown in SEQ ID NO: 71. The amino acid substitution is preferably performed in the light chain variable region of a humanized PM-1 antibody.

<FR and CDR of H53 / L28>
The present invention also provides anti-human IL-6 receptor antibodies with improved plasma pharmacokinetics, stability and / or immunogenicity. In IgG, it has been found that the plasma half-life of IgG having the same Fc region correlates with pI with a high correlation coefficient. Thus, when two types of antibodies against different antigens were tried to modify the pI of the variable region, the plasma half-life was successfully controlled without modifying the Fc region regardless of the type of antigen. The rate of nonspecific uptake of antibodies into endothelial cells is thought to depend on the physicochemical Coulomb interaction between IgG and the negatively charged cell surface. Lowering the IgG pI reduces Coulomb interactions and reduces nonspecific uptake into endothelial cells, resulting in increased plasma retention by reducing metabolism in endothelial cells Become.

  That is, the present invention relates to an anti-IL-6 receptor antibody, particularly an anti-human IL-6 receptor antibody having a reduced isoelectric point and increased plasma retention by replacing the amino acid sequence of a humanized PM-1 antibody. I will provide a. Specifically, in Kabat numbering (Kabat EA et al., 1991 Sequences of Proteins of Immunological Interest. NIH), H13 (13th amino acid of SEQ ID NO: 7) and H16 (SEQ ID NO: 7) of humanized PM-1 7 (16th amino acid), H43 (8th amino acid of SEQ ID NO: 8), H81 (16th amino acid of SEQ ID NO: 9), H105 (3rd amino acid of SEQ ID NO: 10), L18 (sequence) No. 11: 18th amino acid), L45 (11th amino acid of SEQ ID NO: 12), L79 (23rd amino acid of SEQ ID NO: 13), L107 (10th amino acid of SEQ ID NO: 14), H31 (The first amino acid of SEQ ID NO: 1), L24 (the first amino acid of SEQ ID NO: 4) and / or L53 (the fourth amino acid of SEQ ID NO: 5) to another amino acid whose isoelectric point decreases Replace. This makes it possible to reduce the isoelectric point without affecting the binding activity and stability of humanized PM-1. Further, in the humanized PM-1 antibody, when the mouse sequence is humanized, some amino acid residues are left in the mouse sequence in order to retain the binding activity. Specifically, in the Kabat numbering described above, H27 (the 27th amino acid of SEQ ID NO: 7), H28 (the 28th amino acid of SEQ ID NO: 7), H29 (SEQ ID NO: 7) in the humanized PM-1 antibody. The mouse sequence is used as it is for H30 (the 30th amino acid of SEQ ID NO: 7) and H71. HFR1 can be converted to human sequence as HFR1 by substituting H13, H16, H23, and H30, and it is possible to produce an antibody with a further reduced immunogenicity risk than humanized antibody PM-1. Conceivable. Furthermore, since humanized PM-1 is an antibody that has been humanized by CDR grafting, there is room for improvement in terms of stability. For example, it is considered that the antibody can be stabilized by substituting a hydrophilic amino acid for an amino acid residue exposed on the surface in the variable region of the antibody. Furthermore, the antibody can be stabilized by modifying the CDR sequence into a consensus sequence. In the humanized PM-1 antibody, in the Kabat numbering described above, substitution of H69 (the fourth amino acid of SEQ ID NO: 9) from Met to Ile (stabilization of the hydrophobic core structure), H70 (SEQ ID NO: 9) Substitution of Leu to Ser (5th amino acid) (hydrophilization of surface exposed residue), Thr to Asn of H58 (9th amino acid of SEQ ID NO: 2) (consensus sequence of heavy chain CDR2) Modification), substitution of H65 (the 16th amino acid of SEQ ID NO: 2) from Ser to Gly (substitution of Gly to the β-turn portion, modification to the consensus sequence of heavy chain CDR2), or L93 (SEQ ID NO: It is possible to stabilize the antibody by substitution of Thr to Ser (5th amino acid of 6) (hydrophilization of surface exposed residues). In addition, the T-cell epitope predicted in silico can be removed without affecting the binding activity or stability by replacing Thr of L51, the second of the above-mentioned LCDR2 (SEQ ID NO: 5), with Gly. Can reduce the risk of immunogenicity. By combining these amino acid substitutions, it is possible to obtain an anti-IL-6 receptor antibody with improved plasma pharmacokinetics, immunogenicity, and stability of the antibody.

Examples of such antibodies include the antibodies described in any of (1) to (37) below.
(1) An antibody comprising a heavy chain variable region having FR1 in which the 13th Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 7.
The amino acid sequence after substitution is not particularly limited, but substitution with Lys is preferred.
The sequence in which the 13th Arg is substituted with Lys in the amino acid sequence set forth in SEQ ID NO: 7 is shown in SEQ ID NO: 80.
(2) An antibody comprising a heavy chain variable region having FR1 in which the 16th Gln is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 7.
The amino acid sequence after substitution is not particularly limited, but substitution to Glu is preferred.
A sequence in which the 16th Gln is substituted with Glu in the amino acid sequence shown in SEQ ID NO: 7 is shown in SEQ ID NO: 81.
(3) An antibody comprising a heavy chain variable region having FR1 in which the 23rd Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 7.
The amino acid sequence after substitution is not particularly limited, but substitution to Ala is preferred.
The sequence in which the 23rd Thr is substituted with Ala in the amino acid sequence set forth in SEQ ID NO: 7 is shown in SEQ ID NO: 82.
(4) An antibody comprising a heavy chain variable region having FR1 in which the 30th Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 7.
The amino acid sequence after substitution is not particularly limited, but substitution with Ser is preferable.
In the amino acid sequence described in SEQ ID NO: 7, the sequence in which the 30th Thr is replaced with Ser is shown in SEQ ID NO: 83.
(5) An antibody comprising a heavy chain variable region having FR1 in which the 13th Arg, 16th Gln, 23rd Thr and 30th Thr are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 7 .
The amino acid after substitution is not particularly limited, but substitution of Lys for the 13th Arg, Glu for the 16th Gln, Ala for the 23rd Thr, and Ser for the 30th Thr is preferable.
The sequence in which the 13th Arg is replaced with Lys, the 16th Gln is replaced with Glu, the 23rd Thr is replaced with Ala, and the 30th Thr is replaced with Ser in the amino acid sequence shown in SEQ ID NO: 7 is shown in SEQ ID NO: 84
(6) An antibody comprising a heavy chain variable region having FR2 in which the 8th Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 8.
The amino acid after substitution is not particularly limited, but substitution to Glu is preferred.
A sequence in which the 8th Arg is substituted with Glu in the amino acid sequence shown in SEQ ID NO: 8 is shown in SEQ ID NO: 85.
(7) An antibody comprising a heavy chain variable region having FR3 in which the fourth Met is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 9.
The amino acid after substitution is not particularly limited, but substitution to Ile is preferred.
The sequence in which the fourth Met is substituted with Ile in the amino acid sequence set forth in SEQ ID NO: 9 is shown in SEQ ID NO: 86.
(8) An antibody comprising a heavy chain variable region having FR3 in which the fifth Leu is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 9.
The amino acid sequence after substitution is not particularly limited, but substitution with Ser is preferable.
In the amino acid sequence shown in SEQ ID NO: 9, the sequence in which the fifth Leu is replaced with Ser is shown in SEQ ID NO: 87.
(9) An antibody comprising a heavy chain variable region having FR3 in which the 16th Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 9.
The amino acid after substitution is not particularly limited, but substitution with Lys is preferable.
A sequence in which the 16th Arg is substituted with Lys in the amino acid sequence shown in SEQ ID NO: 9 is shown in SEQ ID NO: 88.
(10) An antibody comprising a heavy chain variable region having FR3 in which the 27th Val is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 9.
The amino acid sequence after substitution is not particularly limited, but substitution to Ala is preferred.
A sequence in which the 27th Val is substituted with Ala in the amino acid sequence set forth in SEQ ID NO: 9 is shown in SEQ ID NO: 89.
(11) A heavy chain variable region having FR3 in which the fourth Met, the fifth Leu, the 16th Arg and the 27th Val are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 9 described in (HFR3) An antibody comprising
The amino acid after the substitution is not particularly limited, but the 4th Met is preferably replaced with Ile, the 5th Leu with Ser, the 16th Arg with Lys, and the 27th Val with Ala.
In the amino acid sequence shown in SEQ ID NO: 9, the sequence in which the fourth Met is replaced with Ile, the fifth Leu is replaced with Ser, the 16th Arg is replaced with Lys, and the 27th Val is replaced with Ala is represented by SEQ ID NO: 90.
(12) An antibody comprising a heavy chain variable region having FR4 in which the third Gln is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 10 (HFR4).
The amino acid after substitution is not particularly limited, but substitution to Glu is preferred.
The sequence in which the third Gln is substituted with Glu in the amino acid sequence set forth in SEQ ID NO: 10 is shown in SEQ ID NO: 91.
(13) An antibody comprising a light chain variable region having FR1 in which the 18th Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 11 (LFR1).
The amino acid after substitution is not particularly limited, but substitution to Ser is preferable.
A sequence in which the 18th Arg is substituted with Ser in the amino acid sequence shown in SEQ ID NO: 11 is shown in SEQ ID NO: 92.
(14) An antibody comprising a light chain variable region having FR2 in which the 11th Lys is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 12 (LFR2).
The amino acid after substitution is not particularly limited, but substitution to Glu is preferred.
The sequence in which the 11th Lys in the amino acid sequence set forth in SEQ ID NO: 12 is replaced with Glu is shown in SEQ ID NO: 93.
(15) An antibody comprising a light chain variable region having FR3 in which the 23rd Gln is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 13.
The amino acid after substitution is not particularly limited, but substitution to Glu is preferred.
The sequence in which the 23rd Gln is substituted with Glu in the amino acid sequence set forth in SEQ ID NO: 13 is shown in SEQ ID NO: 94.
(16) An antibody comprising a light chain variable region having FR3 in which the 24th Pro in the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid.
The amino acid sequence after substitution is not particularly limited, but substitution to Ala is preferred.
The sequence in which the 24th Pro in the amino acid sequence described in SEQ ID NO: 13 is substituted with Ala is shown in SEQ ID NO: 95.
(17) An antibody comprising a light chain variable region having FR3 in which the 27th Ile is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 13.
The amino acid sequence after substitution is not particularly limited, but substitution to Ala is preferred.
The sequence in which the 27th Ile is replaced with Ala in the amino acid sequence set forth in SEQ ID NO: 13 is shown in SEQ ID NO: 96.
(18) An antibody comprising a light chain variable region having FR3 in which the 23rd Gln, the 24th Pro and the 27th Ile are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 13 (LFR3).
The amino acid after substitution is not particularly limited, but the 23rd Gln is preferably Glu, the 24th Pro is Ala, and the 27th Ile is preferably Ala.
In the amino acid sequence shown in SEQ ID NO: 13, the sequence in which the 23rd Gln is replaced with Glu, the 24th Pro is Ala, and the 27th Ile is replaced with Ala is shown in SEQ ID NO: 97.
(19) An antibody comprising a light chain variable region having FR4 in which the 10th Lys is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 14 described in (LFR4).
The amino acid after substitution is not particularly limited, but substitution to Glu is preferred.
The sequence in which the 10th Lys is substituted with Glu in the amino acid sequence set forth in SEQ ID NO: 14 is shown in SEQ ID NO: 98.
(20) An antibody comprising a heavy chain variable region having FR4 in which the fifth Ser is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 10 (HFR4).
The amino acid after substitution is not particularly limited, but substitution with Thr is preferable.
In the amino acid sequence shown in SEQ ID NO: 10, the sequence in which the 5th Ser is replaced with Thr is shown in SEQ ID NO: 132.
(21) An antibody comprising a heavy chain variable region having FR4 in which the third Gln and the fifth Ser are substituted with other amino acids in the amino acid sequence of SEQ ID NO: 10 (HFR4).
The amino acid after the substitution is not particularly limited, but substitution of Glu for the third Gln and Thr for the fifth Ser is preferable.
In the amino acid sequence shown in SEQ ID NO: 10, the sequence in which the third Gln is replaced with Glu and the fifth Ser is replaced with Thr is shown in SEQ ID NO: 133.
(22) An antibody comprising a humanized PM-1 heavy chain variable region in which the amino acid substitution according to (5), (6), (11) and (21) has been performed.
(23) An antibody comprising a humanized PM-1 light chain variable region in which the amino acid substitution described in (13), (14), (18) and (19) has been performed.
(24) An antibody comprising the heavy chain variable region described in (22) and the light chain variable region described in (23).
(25) An antibody comprising a heavy chain variable region having CDR1 in which the first Ser is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 1 (HCDR1).
The amino acid after substitution is not particularly limited, but substitution to Asp is preferable.
The sequence in which the first Ser is replaced with Asp in the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 28.
(26) An antibody comprising a heavy chain variable region having CDR2 in which the 16th Ser in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid.
The amino acid after substitution is not particularly limited, but substitution with Gly is preferred.
The sequence in which the 16th Ser in the amino acid sequence described in SEQ ID NO: 2 is replaced with Gly is shown in SEQ ID NO: 99.
(27) An antibody comprising a heavy chain variable region having CDR2 in which the 9th Thr and 16th Ser are substituted with other amino acids in the amino acid sequence described in SEQ ID NO: 2 (HCDR2).
The amino acid after substitution is not particularly limited, but substitution of 9th Thr to Asn and 16th Ser to Gly is preferred.
In the amino acid sequence shown in SEQ ID NO: 2, the sequence in which the 9th Thr is replaced with Asn and the 16th Ser is replaced with Gly is shown in SEQ ID NO: 100.
(28) An antibody comprising a light chain variable region having CDR1 in which the first Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 4 described in (LCDR1).
The amino acid after substitution is not particularly limited, but substitution to Gln is preferable.
The sequence in which the first Arg is substituted with Gln in the amino acid sequence described in SEQ ID NO: 4 is shown in SEQ ID NO: 101.
(29) An antibody comprising a light chain variable region having CDR2 in which the fourth Arg is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 5.
The amino acid after substitution is not particularly limited, but substitution to Glu is preferred.
A sequence in which the 4th Arg is substituted with Glu in the amino acid sequence shown in SEQ ID NO: 5 is shown in SEQ ID NO: 102.
(30) An antibody comprising a light chain variable region having CDR2 in which the second Thr and the fourth Arg are substituted with other amino acids in the amino acid sequence described in SEQ ID NO: 5 (LCDR2).
The amino acid after the substitution is not particularly limited, but substitution of Gr for the second Thr and Glu for the fourth Arg is preferable.
In the amino acid sequence shown in SEQ ID NO: 5, the sequence in which the second Thr is replaced with Gly and the fourth Arg is replaced with Glu is shown in SEQ ID NO: 103.
(31) An antibody comprising a light chain variable region having CDR3 in which the fifth Thr is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 6 (LCDR3).
The amino acid after substitution is not particularly limited, but substitution to Ser is preferable.
In the amino acid sequence shown in SEQ ID NO: 6, the sequence in which the 5th Thr is replaced with Ser is shown in SEQ ID NO: 77.
(32) An antibody comprising a heavy chain variable region in which the amino acid substitution according to (25) and (27) has been performed.
(33) An antibody comprising a light chain variable region having the amino acid substitution described in (28), (30) and (31).
(34) An antibody comprising the heavy chain variable region according to (32) and the light chain variable region according to (33).
(35) An antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 104 (VH of H53 / L28).
(36) An antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 105 (VL of H53 / L28).
(37) An antibody having the heavy chain variable region according to (35) and the light chain variable region according to (36).

The amino acid substitution described in any of (1) to (37) above is preferably performed on the humanized PM-1 antibody. The present invention provides an antibody comprising at least the amino acid substitution according to any one of the above (1) to (37) and a method for producing the antibody. Therefore, the antibody of the present invention includes antibodies containing amino acid substitutions other than the amino acid substitutions described in the above (1) to (37) in addition to the amino acid substitutions described in any of the above (1) to (37). included. The antibody of the present invention also includes an antibody in which a plurality of amino acid substitutions described in any of (1) to (37) above are combined. Examples of the amino acid substitution described in the above (1) to (37) include substitution of the above-described FR and CDR amino acid sequences. Examples of amino acid substitutions other than the amino acid substitutions described in (1) to (37) above include substitutions, deletions, additions and / or insertions of FR and CDR sequences other than those described above. Moreover, substitution, deletion, addition and / or insertion of the amino acid sequence of the constant region can be mentioned.
Furthermore, other than the amino acid modifications described above, examples of modifications that reduce the isoelectric point without reducing the activity of the anti-IL-6 receptor antibody include, for example, the 15th Lys and / or the amino acid sequence shown in SEQ ID NO: 2. Alternatively, modification in which the 16th Ser is substituted with another amino acid can be mentioned. The amino acid after substitution is not particularly limited, but it is preferred that the 15th Lys is substituted with Gln and the 16th Ser is substituted with Asp. In the amino acid sequence of SEQ ID NO: 2, the sequence in which the 15th Lys is replaced with Gln and the 16th Ser is replaced with Asp is shown in SEQ ID NO: 121. Such amino acid substitution may be performed on the amino acid sequence set forth in SEQ ID NO: 100. In the amino acid sequence of SEQ ID NO: 100, the sequence in which the 15th Lys is replaced with Gln and the 16th Gly is replaced with Asp is shown in SEQ ID NO: 122. Accordingly, the present invention provides an antibody comprising a heavy chain variable region having CDR2 in which the 15th Lys and / or the 16th Ser is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 100.
Furthermore, other modifications that lower the isoelectric point include modifications that substitute the 4th Gln with another amino acid in the amino acid sequence set forth in SEQ ID NO: 4. The amino acid after substitution is not particularly limited, but substitution with Glu is preferred. The amino acid sequence in which the fourth Gln is substituted with Glu in the amino acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 123. This amino acid substitution may be performed on the amino acid sequence of SEQ ID NO: 101. The amino acid sequence in which the fourth Gln is substituted with Glu in the amino acid sequence of SEQ ID NO: 101 is shown in SEQ ID NO: 124. Accordingly, the present invention provides an antibody comprising a light chain variable region having CDR1 in which the fourth Gln is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 101.
Furthermore, as another modification that lowers the isoelectric point, a modification in which the 6th His in the amino acid sequence shown in SEQ ID NO: 5 is substituted with another amino acid can be mentioned. The amino acid after substitution is not particularly limited, but substitution with Glu is preferred. The amino acid sequence in which the sixth His is substituted with Glu in the amino acid sequence set forth in SEQ ID NO: 5 is shown in SEQ ID NO: 125. This amino acid substitution may be performed on the amino acid sequence of SEQ ID NO: 103. The amino acid sequence in which the 6th His is replaced with Glu in the amino acid sequence of SEQ ID NO: 103 is shown in SEQ ID NO: 126. Accordingly, the present invention provides an antibody comprising a light chain variable region having CDR2 in which the 6th His is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103.
Furthermore, in the amino acid sequence of heavy chain FR3 set forth in SEQ ID NO: 90, modifications that reduce the immunogenicity risk include a modification in which Ala at the 27th position (Kabat numbering H89) is replaced with Val. The amino acid sequence in which the 27th Ala is substituted with Val in the amino acid sequence set forth in SEQ ID NO: 90 is shown in SEQ ID NO: 127. Therefore, the present invention provides an antibody comprising a heavy chain variable region having FR3 in which the 27th Ala is substituted with Val in the amino acid sequence of SEQ ID NO: 90.
The human VH1 subclass in which H71 is conserved in Arg with respect to Arg at position 6 (Kabat numbering H71), the only remaining mouse sequence in the amino acid sequence of heavy chain FR3 set forth in SEQ ID NO: 9 or SEQ ID NO: 90 (SEQ ID NO: 128) Alternatively, human sequences of human VH3 subclass (SEQ ID NO: 129) can be used as FR3 sequences to produce anti-human IL-6 receptor antibodies that are completely human sequences. It is believed that there is. Accordingly, the present invention provides an antibody comprising a heavy chain variable region having the FR3 set forth in SEQ ID NO: 128 or SEQ ID NO: 129.
Furthermore, in the amino acid sequence of heavy chain FR4 shown in SEQ ID NO: 10, a modification that improves the stability can be exemplified by a substitution of Ser at the fifth position (Kabat numbering H107) with Ile. In the amino acid sequence shown in SEQ ID NO: 10, the amino acid sequence in which the 5th Ser is replaced with Ile is shown in SEQ ID NO: 130. This amino acid sequence may be performed on the amino acid sequence of SEQ ID NO: 91. In the amino acid sequence set forth in SEQ ID NO: 91, the amino acid sequence in which the 5th Ser is replaced with Ile is shown in SEQ ID NO: 131. Accordingly, the present invention provides an antibody comprising a heavy chain variable region having FR4 in which the 5th Ser is replaced with Ile in the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 91.
Such amino acid substitution is performed by humanized PM-1 antibody, H53 / L28 (an antibody including the heavy chain variable region of SEQ ID NO: 104, the light chain variable region of SEQ ID NO: 105), or PF1 antibody (SEQ ID NO: 22 It is preferable to carry out the treatment on a heavy chain variable region, an antibody comprising the light chain variable region of SEQ ID NO: 23). The present invention provides an antibody containing at least such an amino acid substitution and a method for producing the antibody. Therefore, in addition to such amino acid substitutions, the antibodies of the present invention include antibodies comprising the amino acid substitutions described in the above (1) to (37) and / or amino acid substitutions other than the above (1) to (37). included. Examples of amino acid substitutions other than the amino acid substitutions described in (1) to (37) above include substitutions, deletions, additions and / or insertions of FR and CDR sequences other than those described above. Moreover, substitution, deletion, addition and / or insertion of the amino acid sequence of the constant region can be mentioned.

<Anti-human IL-6 receptor antibody with low isoelectric point>
The present invention further provides an anti-IL-6 receptor antibody having a low isoelectric point. Antibodies with a low isoelectric point of the present invention include antibodies with a low measured isoelectric point of full length antibodies and antibodies with a low theoretical isoelectric point of variable regions (VH / VL).
In the present invention, the anti-IL-6 receptor antibody having a low measured isoelectric point of the full-length antibody is usually an antibody having a measured isoelectric point of 7.5 or less, preferably an antibody having a measured isoelectric point of 7.0 or less, Preferably, the antibody has a measured isoelectric point of 6.0 or less. The measured isoelectric point can be measured by a method known to those skilled in the art. For example, it can be measured by a method such as non-denaturing gel isoelectric focusing or capillary isoelectric focusing.
In the present invention, the anti-IL-6 receptor antibody having a low theoretical isoelectric point of the variable region is usually an antibody having a theoretical isoelectric point of 5.5 or less, preferably an antibody having a theoretical isoelectric point of 5.0 or less, Preferably, the antibody has a theoretical isoelectric point of 4.0 or less. The theoretical isoelectric point can be calculated by a method known to those skilled in the art.For example, the theoretical isoelectric point of the variable region VH and VL can be calculated by using software such as GENETYX (GENETYX CORPORATION). Is possible.
In the anti-IL-6 receptor antibody having a low isoelectric point of the present invention, the amino acid substitution introduced is not particularly limited, and examples thereof include the amino acid substitution described above. Such an anti-IL-6 receptor antibody having a low isoelectric point is considered to have improved retention in plasma.
The IL-6 receptor is not particularly limited, but human IL-6 receptor is preferred.

<Stable anti-human IL-6 receptor antibody at high concentration>
Furthermore, the present invention provides anti-IL-6 receptor antibodies that are stable at high concentrations.
In the present invention, “stable at a high concentration” means that anti-IL− in appropriately selected buffer conditions (for example, 20 mM histidine-HCl, 150 mM NaCl) within a pH range of 6.5 to 7.0 suitable for subcutaneous administration. Increase of the aggregate ratio (aggregate peak area on gel filtration chromatography / total peak area × 100) per month of the high-concentration antibody solution of 6 receptor antibody 100 mg / mL at 25 ° C. is preferably 0.3% or less, preferably It means 0.2% or less, more preferably 0.1% or less. The concentration of the anti-IL-6 receptor antibody may be 100 mg / mL or more, for example, 200 mg / mL or 300 mg / mL.
The anti-IL-6 receptor antibody stable at a high concentration of the present invention is not particularly limited, but can be prepared by, for example, the above-described amino acid substitution.
The IL-6 receptor is not particularly limited, but human IL-6 receptor is preferred.

  The present invention also provides a humanized PM-1 antibody in which the amino acid substitution according to any one of the above (1) to (37) is performed, and further according to any one of the above (a) to (y). An antibody having an amino acid substitution that improves the binding activity and / or neutralization activity is provided. One embodiment of such an antibody has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 22 (PF1_H) and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 (PF1_L). An antibody (PF1) can be mentioned, but is not limited thereto.

Furthermore, the present invention provides the anti-IL-6 receptor antibody described in any of the following antibodies (A) to (I).
(A) CDR1 having the amino acid sequence described in SEQ ID NO: 165 (CDR1 of VH5-M83), CDR2 having the amino acid sequence described in SEQ ID NO: 166 (CDR2 of VH5-M83), described in SEQ ID NO: 167 A heavy chain variable region having CDR3 having an amino acid sequence (CDR3 of VH5-M83);
(B) CDR1 having the amino acid sequence described in SEQ ID NO: 101 (CDR1 of VL5), CDR2 having the amino acid sequence described in SEQ ID NO: 168 (CDR2 of VL5), amino acid sequence described in SEQ ID NO: 79 (VL5 A light chain variable region having CDR3,
(C) an antibody comprising the heavy chain variable region of (A) and the light chain variable region of (B),
(D) CDR1 having the amino acid sequence described in SEQ ID NO: 169 (CDR1 of VH3-M73), CDR2 having the amino acid sequence described in SEQ ID NO: 170 (CDR2 of VH3-M73), described in SEQ ID NO: 171 A heavy chain variable region having CDR3 having an amino acid sequence (CDR3 of VH3-M73);
(E) CDR1 having the amino acid sequence described in SEQ ID NO: 172 (CDR1 of VL3), CDR2 having the amino acid sequence described in SEQ ID NO: 173 (CDR2 of VL3), amino acid sequence described in SEQ ID NO: 79 (VL3 A light chain variable region having CDR3,
(F) an antibody comprising the heavy chain variable region of (D) and the light chain variable region of (E),
(G) CDR1 having the amino acid sequence described in SEQ ID NO: 169 (CDR1 of VH4-M73), CDR2 having the amino acid sequence described in SEQ ID NO: 174 (CDR2 of VH4-M73), described in SEQ ID NO: 171 A heavy chain variable region having CDR3 having an amino acid sequence (CDR3 of VH4-M73);
(H) CDR1 having the amino acid sequence described in SEQ ID NO: 175 (CDR1 of VL1), CDR2 having the amino acid sequence described in SEQ ID NO: 173 (CDR2 of VL1), amino acid sequence described in SEQ ID NO: 79 (VL1 A light chain variable region having CDR3,
(I) An antibody comprising the heavy chain variable region of (G) and the light chain variable region of (H).

Furthermore, the present invention provides the anti-IL-6 receptor antibody described in any of (a) to (q) below.
(a) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 159 (H96-IgG1 variable region),
(b) in the amino acid sequence shown in SEQ ID NO: 159 (H96-IgG1 variable region), 35th Trp, 51st Tyr, 63rd Ser, 65th Lys, 66th Gly, 99th Val An antibody comprising a heavy chain variable region having an amino acid sequence in which at least one amino acid of the 103rd Ile, the 108th Tyr, the 111th Glu, and the 113th Thr is substituted with another amino acid,
(c) In the amino acid sequence described in SEQ ID NO: 159 (H96-IgG1 variable region), 65th Lys, 66th Gly, 99th Val, 103rd Ile, 111th Glu, 113th Thr An antibody comprising a heavy chain variable region having an amino acid sequence substituted with another amino acid,
(d) in the amino acid sequence shown in SEQ ID NO: 159 (H96-IgG1 variable region), 35th Trp, 51st Tyr, 63rd Ser, 65th Lys, 66th Gly, 99th Val An antibody comprising a heavy chain variable region having an amino acid sequence in which the 103rd Ile, 108th Tyr is substituted with another amino acid,
(e) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 160 (F2H-IgG1 variable region),
(f) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 161 (VH5-M83 variable region),
(g) an antibody having a light chain variable region having an amino acid sequence in which the 27th Gln and / or the 55th His is substituted with another amino acid in the amino acid sequence (PF1L) described in SEQ ID NO: 23;
(h) an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 162 (L39 variable region),
(i) an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 163 (VL5-kappa variable region),
(j) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 176 (VH3-M73 variable region),
(k) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 178 (VH4-M73 variable region),
(l) an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 177 (VL3-kappa variable region),
(m) an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 179 (VL1-kappa variable region),
(n) an antibody comprising the heavy chain variable region of (e) and the light chain variable region of (h),
(o) an antibody comprising the heavy chain variable region of (f) and the light chain variable region of (i) (FV5-M83 variable region combination),
(p) an antibody comprising the heavy chain variable region of (j) and the light chain variable region of (l) (FV4-M73 variable region combination),
(q) An antibody (a variable region combination of FV3-M73) comprising the heavy chain variable region of (k) and the light chain variable region of (m).

  In the amino acid substitution of the heavy chain variable region of (a) to (d) described above, the amino acid after substitution is not particularly limited, but the 35th Trp is Val, the 51st Tyr is Phe, the 63rd Ser is Thr, 65th Lys is Gln, 66th Gly is Asp, 99th Val is Leu, 103th Ile is Ala, 108th Tyr is Val, 111th Glu is Gln, 113th Thr is Ile It is preferred that In the amino acid substitution in the light chain variable region of (g) described above, the amino acid after substitution is not particularly limited, but it is preferred that the 27th Gln is substituted with Glu and the 55th His is substituted with Glu. In addition, amino acid substitutions, deletions, insertions, and / or additions other than those described above may be performed.

The constant region of the antibody of the present invention is not particularly limited, and any constant region may be used. For example, a constant region having a natural sequence such as IgG1, IgG2, IgG4, or a modified constant region prepared by performing substitution, deletion, addition and / or insertion of an amino acid in a constant region having a natural sequence Etc. can be used. Examples of the modified constant region include the constant regions described later.
Examples of the antibody using the variable region of the present invention described above include the following antibodies.
(1) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 134 (H96-IgG1),
(2) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 135 (F2H-IgG1),
(3) an antibody comprising a heavy chain having the amino acid sequence (VH5-IgG1) set forth in SEQ ID NO: 137,
(4) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 139 (VH5-M83),
(5) an antibody comprising a light chain having the amino acid sequence (L39) set forth in SEQ ID NO: 136,
(6) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 138 (VL5-kappa),
(7) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 180 (VH3-M73),
(8) an antibody comprising a heavy chain having the amino acid sequence (VH4-M73) set forth in SEQ ID NO: 182;
(9) an antibody comprising a light chain having the amino acid sequence (VL3-kappa) set forth in SEQ ID NO: 181;
(10) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 183 (VL1-kappa),
(11) an antibody comprising the heavy chain of (2) and the light chain of (5),
(12) an antibody comprising the heavy chain of (3) and the light chain of (6),
(13) an antibody (FV5-M83) comprising the heavy chain of (4) and the light chain of (6),
(14) an antibody (FV4-M73) comprising the heavy chain of (7) and the light chain of (9),
(15) an antibody (FV3-M73) comprising the heavy chain of (8) and the light chain of (10),
(16) An antibody having an activity equivalent to that of the antibody according to any one of (1) to (15).
Here, “having equivalent activity” means that the antigen-binding activity and / or neutralizing activity is equivalent. In the present invention, the equivalent activity is not necessarily the same activity, and means, for example, having 50% or more activity, preferably 70% or more activity, more preferably 90% or more activity.

Furthermore, the present invention provides the CDR or FR described in any of the following (i) to (xxii).
(i) Heavy chain FR1 having the amino acid sequence set forth in SEQ ID NO: 84 (VH5 heavy chain FR1)
(ii) Heavy chain FR1 having the amino acid sequence set forth in SEQ ID NO: 186 (VH3, VH4 heavy chain FR1)
(iii) Heavy chain FR2 having the amino acid sequence set forth in SEQ ID NO: 85 (VH3, VH4, VH5 heavy chain FR2)
(iv) Heavy chain FR3 having the amino acid sequence set forth in SEQ ID NO: 184 (VH3, VH4, VH5 heavy chain FR3)
(v) Heavy chain FR4 having the amino acid sequence set forth in SEQ ID NO: 133 (heavy chain FR4 of VH3, VH4, VH5)
(vi) Light chain FR1 having the amino acid sequence set forth in SEQ ID NO: 92 (VL1, VL3, VL5 light chain FR1)
(vii) Light chain FR2 having the amino acid sequence set forth in SEQ ID NO: 93 (light chain FR2 of VL1, VL3, VL5)
(viii) Light chain FR3 having the amino acid sequence of SEQ ID NO: 97 (light chain FR3 of VL1, VL3, VL5)
(ix) Light chain FR4 having the amino acid sequence set forth in SEQ ID NO: 98 (VL1, VL3, VL5 light chain FR4)
(x) heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 169 (heavy chain CDR1 of VH3, VH4)
(xi) heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 165 (heavy chain CDR1 of VH5),
(xii) heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 170 (heavy chain CDR2 of VH3),
(xiii) a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 174 (heavy chain CDR2 of VH4),
(xiv) a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 166 (heavy chain CDR2 of VH5),
(xv) heavy chain CDR3 (VH3, heavy chain CDR3 of VH4) having the amino acid sequence of SEQ ID NO: 171,
(xvi) a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 167 (heavy chain CDR3 of VH5),
(xvii) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 175 (light chain CDR1 of VL1),
(xviii) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 172 (light chain CDR1 of VL3),
(xix) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 101 (a light chain CDR1 of VL5),
(xx) a light chain CDR2 (VL1, VL3 light chain CDR2) having the amino acid sequence set forth in SEQ ID NO: 173,
(xxi) a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 168 (light chain CDR2 of VL5),
(xxii) Light chain CDR3 having the amino acid sequence of SEQ ID NO: 79 (VL1, VL3, VL5 light chain CDR3)

In addition, the antibody of the present invention includes antibody fragments containing any of the amino acid substitutions described above and modified products thereof. For example, antibody fragments include Fab, F (ab ') 2, Fv, or single chain Fv (scFv) in which H chain and L chain Fv are linked by an appropriate linker, H chain single domain or L chain single domain. (For example, Nat Biotechnol. 2005 Sep; 23 (9): 1126-36.), Unibody (WO2007059782 A1), SMIP (WO2007014278 A2). The origin of the antibody is not particularly limited, and examples thereof include a human antibody, a mouse antibody, a rat antibody, and a rabbit antibody. The antibody of the present invention may be a chimeric antibody, a humanized antibody, a fully humanized antibody or the like.
Specifically, the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or a gene encoding these antibody fragments is constructed and introduced into an expression vector, and then an appropriate host cell. (E.g., Co, MS et al., J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Pluckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 497-515, Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-66, Bird, RE et al., TIBTECH (1991) 9, 132-137).

  scFv is obtained by linking the H chain V region and L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879. -5883). The H chain V region and the L chain V region in scFv may be derived from any of those described as the above antibody. As the peptide linker that links the V regions, for example, any single chain peptide consisting of amino acid residues 12-19 is used.

<Antibody constant region>
The present invention also provides an antibody constant region improved by substituting the amino acids described in any of (i) to (xxi) below. The constant region means an IgG1, IgG2, or IgG4 type constant region. The amino acid sequences of human IgG1 constant region, human IgG2 constant region and human IgG4 constant region are known (human IgG1 constant region: SEQ ID NO: 19, human IgG2 constant region: SEQ ID NO: 20, human IgG4 constant region: SEQ ID NO: 21). The human IgG4 constant region is a sequence into which a modification (Mol Immunol. 1993 Jan; 30 (1): 105-8.) For improving the stability of the hinge portion is introduced. The present invention further provides an antibody comprising an antibody constant region in which the amino acid is substituted. The antibody constant region is preferably a human antibody constant region.
The antibody constant region substituted with the amino acid of the present invention may contain other amino acid substitutions and modifications as long as it contains the amino acid substitution described in any of (i) to (xxi) below. Therefore, in the present invention, when the amino acid substitution of the present invention is performed on an IgG2 constant region in which one or more amino acids have already been substituted and / or modified from the amino acid sequence shown in SEQ ID NO: 20, or Even when one or a plurality of amino acids are substituted and / or modified after amino acid substitution, the IgG2 constant region having the amino acid sequence of SEQ ID NO: 20 corresponds to the IgG2 constant region in which the amino acid substitution of the present invention has been performed. To do. The same applies to the IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19 and the IgG4 constant region set forth in SEQ ID NO: 21.
In addition, the 297th sugar chain of EU numbering (see Sequences of proteins of immunological interest, NIH Publication No.91-3242) may have any sugar chain structure, and sugar chains may not be bound ( For example, a constant region produced in a host cell to which no sugar chain is added, such as E. coli).

(I) Improving acid stability of IgG2 constant region As an embodiment of the IgG2 constant region substituted with the amino acid of the present invention, the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20 is the An IgG2 constant region in which Met at number 397) is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution to Val is preferable. By substituting the other amino acid for Met at position 276 (EU number 397) in the amino acid sequence set forth in SEQ ID NO: 20, it is possible to improve the stability of the antibody under acidic conditions.

(Ii) Improvement of heterogeneity of IgG2 constant region As one embodiment of the IgG2 constant region substituted with the amino acid of the present invention, the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20 Numbering 131) Cys, 16th (EU numbering 133rd) Arg, and 102nd (EU numbering 219th) Cys are replaced with IgG2 constant regions by other amino acids. The amino acid after substitution is not particularly limited, but the 14th (EU numbering 131) Cys is preferably substituted with Ser, and the 16th (EU numbering 133rd) Arg is preferably substituted with Lys. , 102nd (219th EU numbering) Cys is preferably replaced with Ser (IgG2-SKSC).
By performing these substitutions, heterogeneity derived from the hinge region of IgG2 can be reduced. The IgG2 constant region substituted with the amino acid of the present invention includes an IgG2 constant region substituted with at least one amino acid among the above three types of amino acid substitutions, except for the 14th Cys and the 102nd Cys. It is preferable that the above amino acid is substituted, or that all three types of amino acids are substituted.

(Iii) Reduction of binding of IgG2 constant region to FcγR As one embodiment of the IgG2 constant region substituted with the amino acid of the present invention, in the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, position 209 (EU330) Provides an IgG2 constant region in which Ala is replaced with Ser, 210th (EU331) Pro with Ser, and / or 218th (EU339) Thr with Ala. Although it has already been reported that the substitution of 209th (EU330) Ala and 210th (EU331) Pro can reduce the binding to Fcγ receptor (Eur J Immunol. 1999 Aug; 29 (8 ): 2613-24.) This modification is not preferable from the viewpoint of immunogenicity risk because non-human-derived peptides that can become T-cell epitopes appear. Therefore, by simultaneously replacing 218th (EU339) Thr with Ala, IgG2 can bind to Fcγ receptor using only human-derived peptides as 9-12 amino acids that can become T-cell epitopes. It can be reduced.
In the IgG2 constant region substituted with the amino acid of the present invention, it is sufficient that at least one amino acid is substituted among the above-mentioned three amino acid substitutions, but preferably all the above three amino acids are substituted. It is preferable. Therefore, as a preferred embodiment of the IgG2 constant region in which the amino acid of the present invention is substituted, in the IgG2 constant region having the amino acid sequence of SEQ ID NO: 20, Ala at position 209 (EU330) is substituted with Ser, and position 210 ( An example is an IgG2 constant region in which Pro in EU331) is replaced with Ser and Thr at 218th (EU339) is replaced with Ala.

(Iv) Improvement of C-terminal heterogeneity of IgG2 constant region The present invention relates to Gly and 326th (EU numbering) 325th (EU numbering 446) Gly and 326th (EU numbering) in the IgG2 constant region having the amino acid sequence of SEQ ID NO: 20. An IgG2 constant region lacking Lys of number 447) is provided. By deleting both of these amino acids, it is possible to reduce heterogeneity derived from the H chain C terminus of an antibody for the first time.

(V) Improvement of retention in plasma by modification of IgG2 constant region As one embodiment of the IgG2 constant region substituted with the amino acid of the present invention, in the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, position 147 Examples include IgG2 constant region in which Gn (EU numbering 268th) His, 234th (EU numbering 355th) Arg, 298th (EU numbering 419th) Gln is substituted with another amino acid. These amino acid substitutions can improve the retention of antibodies in plasma. The amino acid after substitution is not particularly limited, but the 147th (EU numbering 268th) His is preferably substituted with Gln, and the 234th (EU numbering 355th) Arg is preferably substituted with Gln. The 298th (EU numbering 419th) Gln is preferably replaced with Glu. The IgG2 constant region in which amino acids of the present invention are substituted includes an IgG2 constant region in which at least one amino acid is substituted among the above three types of amino acid substitutions, but all the above three types of amino acids are substituted. It is preferable.

(Vi) Improvement of acid stability of IgG4 constant region The present invention relates to IgG4 constant region having the amino acid sequence of SEQ ID NO: 21, wherein Arg at position 289 (EU numbering 409) is substituted with another amino acid. Provides an IgG4 constant region. The amino acid after substitution is not particularly limited, but substitution to Lys is preferable. By substituting Arg at 289th position (409th position of EU numbering) with other amino acid in the amino acid sequence set forth in SEQ ID NO: 21, it is possible to improve the stability of the antibody under acidic conditions.

(Vii) Improvement of C-terminal heterogeneity of IgG4 constant region The present invention relates to Gly at position 326 (EU numbering 446) Gly and 327 (EU numbering) in the IgG4 constant region having the amino acid sequence of SEQ ID NO: 21. An IgG4 constant region lacking Lys of number 447) is provided. By deleting both of these amino acids, it is possible to reduce heterogeneity derived from the H chain C terminus of an antibody for the first time.

(Viii) Improvement of C-terminal heterogeneity of IgG1 constant region The present invention relates to IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19, Gly at position 329 (EU numbering 446) Gly and position 330 (EU An IgG1 constant region lacking Lys of number 447) is provided. By deleting both of these amino acids, it is possible to reduce heterogeneity derived from the H chain C terminus of an antibody for the first time.

(Ix)
The present invention provides an IgG1 constant region having an amino acid sequence in which Asn at the 317th (EU numbering 434th) Asn is substituted with another amino acid in the IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19.
The amino acid after substitution is not particularly limited, but substitution to Ala is preferable.

(X)
The present invention relates to the amino acid sequence shown in SEQ ID NO: 20, Ala at position 209 (EU numbering 330), Pro at position 210 (EU numbering 331), Thr at position 218 (EU numbering 339), position 276 EU numbering 397th Met, 14th (EU numbering 131th) Cys, 16th (EU numbering 133th) Arg, 102nd (EU numbering 219th) Cys, 20th (EU numbering 137th) Provides an IgG2 constant region having an amino acid sequence in which the Glu and the 21st (EU numbering 138th) Ser are replaced with other amino acids.
The amino acid after substitution is not particularly limited, but 209th Ala is Ser, 210th Pro is Ser, 218th Thr is Ala, 276th Met is Val, 14th Cys is Ser, The 16th Arg is preferably replaced with Lys, the 102nd Cys with Ser, the 20th Glu with Gly, and the 21st Ser with Gly.

(Xi)
The present invention relates to the amino acid sequence shown in SEQ ID NO: 20, Ala at position 209 (EU numbering 330), Pro at position 210 (EU numbering 331), Thr at position 218 (EU numbering 339), position 276 EU numbering 397th Met, 14th (EU numbering 131th) Cys, 16th (EU numbering 133th) Arg, 102nd (EU numbering 219th) Cys, 20th (EU numbering 137th) Glu and 21st (EU numbering 138th) Ser are substituted with other amino acids, and 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys are deleted. Provides an IgG2 constant region.
The amino acid after substitution is not particularly limited, but 209th Ala is Ser, 210th Pro is Ser, 218th Thr is Ala, 276th Met is Val, 14th Cys is Ser, The 16th Arg is preferably replaced with Lys, the 102nd Cys with Ser, the 20th Glu with Gly, and the 21st Ser with Gly.

(Xii)
The present invention relates to the amino acid sequence set forth in SEQ ID NO: 20, Met at position 276 (EU numbering 397), Cys at position 14 (EU numbering 131), Arg at position 16 (EU numbering 133), position 102 Provided is an IgG2 constant region having an amino acid sequence in which Cys (EU numbering 219), Glu at 20th (EU numbering 137) and Ser at 21st (EU numbering 138) are substituted with other amino acids.
The amino acid after substitution is not particularly limited, but the 276th Met is Val, the 14th Cys is Ser, the 16th Arg is Lys, the 102nd Cys is Ser, the 20th Glu is Gly, The 21st Ser is preferably substituted with Gly.

(Xiii)
The present invention relates to the amino acid sequence set forth in SEQ ID NO: 20, Met at position 276 (EU numbering 397), Cys at position 14 (EU numbering 131), Arg at position 16 (EU numbering 133), position 102 (EU numbering 219th) Cys, 20th (EU numbering 137th) Glu and 21st (EU numbering 138th) Ser are replaced with other amino acids, and 325th (EU numbering 446th) Gly and An IgG2 constant region having an amino acid sequence in which Lys at position 326 (EU numbering 447) is deleted is provided.
The amino acid after substitution is not particularly limited, but the 276th Met is Val, the 14th Cys is Ser, the 16th Arg is Lys, the 102nd Cys is Ser, the 20th Glu is Gly, The 21st Ser is preferably substituted with Gly.

(Xiv)
The present invention relates to the 14th (EU numbering 131st) Cys, the 16th (EU numbering 133rd) Arg, the 102nd (EU numbering 219th) Cys, the 20th amino acid sequence of SEQ ID NO: 20. (EU numbering 137th) Glu, 21st (EU numbering 138th) Ser, 147th (EU numbering 268th) His, 234th (EU numbering 355th) Arg and 298th (EU numbering 419th) Gln is substituted with another amino acid, and an IgG2 constant region having an amino acid sequence in which 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys are deleted is provided.
The amino acid after substitution is not particularly limited, but the 14th Cys is Ser, the 16th Arg is Lys, the 102nd Cys is Ser, the 20th Glu is Gly, the 21st Ser is Gly, It is preferable that the 147th His is replaced with Gln, the 234th Arg is replaced with Gln, and the 298th Gln is replaced with Glu.

(Xv)
The present invention relates to the 14th (EU numbering 131st) Cys, the 16th (EU numbering 133rd) Arg, the 102nd (EU numbering 219th) Cys, the 20th amino acid sequence of SEQ ID NO: 20. EU numbering 137th Glu, 21st (EU numbering 138th) Ser, 147th (EU numbering 268th) His, 234th (EU numbering 355th) Arg, 298th (EU numbering 419th) Amino acid sequence in which Gln of 313 and Asn at position 313 (EU numbering 434) are substituted with other amino acids, and Gly at position 325 (EU numbering 446) and Lys at position 326 (EU numbering 447) are deleted. An IgG2 constant region is provided.
The amino acid after substitution is not particularly limited, but the 14th Cys is Ser, the 16th Arg is Lys, the 102nd Cys is Ser, the 20th Glu is Gly, the 21st Ser is Gly, It is preferable that the 147th His is replaced with Gln, the 234th Arg is replaced with Gln, the 298th Gln is replaced with Glu, and the 313th Asn is replaced with Ala.

(Xvi)
The present invention relates to the 289th (EU numbering 409th) Arg, 14th Cys, 16th Arg, 20th Glu, 21st Ser, 97th Arg in the amino acid sequence set forth in SEQ ID NO: 21. , 100th Ser, 102th Tyr, 103th Gly, 104th Pro and 105th Pro (EU numbering 131,133,137,138,214,217,219,220,221,222th), 113th Glu, 114th Phe and 115th (EU numbering 233,234,235th) Provides an IgG4 constant region having an amino acid sequence in which Leu is substituted with another amino acid and Gly at position 116 (EU numbering 236) is deleted.
The amino acid after substitution is not particularly limited, but Cys at the 14th (EU numbering 131) is Ser, Arg at the 16th (EU numbering 133) is Lys, Glu at the 20th (EU numbering 137) is Gly, 21 The Ser (EU numbering 138) is Gly, the 97th (EU numbering 214) Arg is Thr, the 100th (EU numbering 217) Ser is Arg, and the 102nd (EU numbering 219) Tyr is Ser. , 103rd (EU numbering 220) Gly is Cys, 104th (EU numbering 221) Pro is Val, 105th (EU numbering 222) Pro is Glu, 113th (EU numbering 233) Glu is Pro, 114th (EU numbering 234) Phe to Val, 115th (EU numbering 235) Leu to Ala, 289th (EU numbering 409) Arg to Lys Is preferred.

(Xvii)
The present invention relates to the 289th (EU numbering 409th) Arg, 14th Cys, 16th Arg, 20th Glu, 21st Ser, 97th Arg in the amino acid sequence set forth in SEQ ID NO: 21. , 100th Ser, 102th Tyr, 103th Gly, 104th Pro and 105th Pro (EU numbering 131,133,137,138,214,217,219,220,221,222th), 113th Glu, 114th Phe and 115th Leu (EU numbering 233,234,235) Is substituted with other amino acids and has an amino acid sequence lacking Gly at position 116 (EU numbering 236), Gly at position 326 (EU numbering 446) and Lys at position 327 (EU numbering 447) Provides an IgG4 constant region.
The amino acid after substitution is not particularly limited, but Cys at the 14th (EU numbering 131) is Ser, Arg at the 16th (EU numbering 133) is Lys, Glu at the 20th (EU numbering 137) is Gly, 21 The Ser (EU numbering 138) is Gly, the 97th (EU numbering 214) Arg is Thr, the 100th (EU numbering 217) Ser is Arg, and the 102nd (EU numbering 219) Tyr is Ser. , 103rd (EU numbering 220) Gly is Cys, 104th (EU numbering 221) Pro is Val, 105th (EU numbering 222) Pro is Glu, 113th (EU numbering 233) Glu is Pro, 114th (EU numbering 234) Phe to Val, 115th (EU numbering 235) Leu to Ala, 289th (EU numbering 409) Arg to Lys Is preferred.

(Xviii)
The present invention relates to the IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19, wherein Asn at position 317 (EU numbering 434) is substituted with another amino acid, and position 329 (EU numbering 446). An IgG1 constant region having an amino acid sequence in which Gly and 330th (EU numbering 447) Lys are deleted is provided.
The amino acid after substitution of Asn at the 317th (EU numbering 434th) Asn is not particularly limited, but substitution to Ala is preferred.

(Xix)
Furthermore, the present invention includes the following IgG2 as a preferred embodiment of IgG2 with improved hinge region heterogeneity and / or reduced Fcγ receptor binding activity.
In IgG2 having a constant region consisting of the amino acid sequence set forth in SEQ ID NO: 20, 209th Ala, 210th Pro, 218th Thr, 14th Cys, 16th Arg, 102th Cys, 20th Glu, an antibody in which the 21st Ser is substituted with another amino acid.
The amino acid after substitution is not particularly limited, but the 209th (EU numbering 330) Ala is Ser, the 210th (EU numbering 331) Pro is Ser, the 218th (EU numbering 339) Thr is Ala, the 14th (EU Numbering 131) Cys is Ser, 16th (EU numbering 133) Arg is Lys, 102th (EU numbering 219) Cys is Ser, 20th (EU numbering 137) Glu is Gly, 21st (EU numbering 138) ) Ser is preferably replaced with Gly.
An example of such an IgG2 constant region is an IgG2 constant region having the amino acid sequence of SEQ ID NO: 191 (M86).
Further, as another preferred embodiment of the IgG2 constant region of the present invention, an IgG2 constant region in which the 325th Gly and 326th Lys are further deleted in the above-mentioned IgG2 constant region in order to reduce the C-terminal heterogeneity. Can be mentioned. An example of such an antibody is IgG2 having a constant region consisting of the amino acid sequence of SEQ ID NO: 192 (M86ΔGK).

(Xx)
Furthermore, the present invention includes the following IgG2 constant regions as preferred embodiments of the IgG2 constant region with improved hinge region heterogeneity.
IgG2 constant region in which the 14th Cys, 16th Arg, 102th Cys, 20th Glu and 21st Ser are substituted with other amino acids in the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20. region.
The amino acid after substitution is not particularly limited, but the 14th (EU numbering 131) Cys is Ser, the 16th (EU numbering 133) Arg is Lys, the 102nd (EU numbering 219) Cys is Ser, and the 20th (EU It is preferable that Glu in the numbering 137) is replaced with Gly and Ser in the 21st (EU numbering 138) is replaced with Gly.
An example of such an IgG2 constant region is an IgG2 constant region having the amino acid sequence of SEQ ID NO: 193 (M40).
Another preferred embodiment of the IgG2 constant region of the present invention is an IgG2 constant region in which the 325th Gly and 326th Lys are further deleted in the above-mentioned IgG2 constant region. An example of such an antibody includes an IgG2 constant region having the amino acid sequence of SEQ ID NO: 194 (M40ΔGK).

(Xxi) M14ΔGK, M17ΔGK, M11ΔGK, M31ΔGK, M58, M73, M83, M86ΔGK, M40ΔGK
The present invention also provides an antibody constant region (M14ΔGK) having the amino acid sequence set forth in SEQ ID NO: 24. The present invention further provides an antibody constant region (M17ΔGK) having the amino acid sequence set forth in SEQ ID NO: 116. The present invention further provides an antibody constant region (M11ΔGK) having the amino acid sequence set forth in SEQ ID NO: 25. Furthermore, the present invention provides a constant region (M31ΔGK) having the amino acid sequence set forth in SEQ ID NO: 118. The present invention further provides a constant region (M58) having the amino acid sequence set forth in SEQ ID NO: 151. Furthermore, the present invention provides a constant region having the amino acid sequence set forth in SEQ ID NO: 153 (M73). The present invention further provides a constant region having the amino acid sequence set forth in SEQ ID NO: 164 (M83). The present invention further provides a constant region having the amino acid sequence set forth in SEQ ID NO: 192 (M86ΔGK). The present invention further provides a constant region having the amino acid sequence set forth in SEQ ID NO: 194 (M40ΔGK). These antibody constant regions have reduced Fcγ receptor binding activity, reduced immunogenicity risk, improved stability under acidic conditions, reduced heterogeneity, improved plasma retention and / or IgG1 An optimized antibody constant region with the property of high stability in the formulation compared to the constant region.

The present invention provides an antibody comprising the antibody constant region described in any of (i) to (xxi) above. As long as it has the above-mentioned antibody constant region, the type of antigen, the origin of the antibody, etc. are not limited, and any antibody may be used. Examples of preferable antibodies include antibodies that bind to IL-6 receptor. Moreover, humanized antibodies can be mentioned as examples of other preferable antibodies. As an example of such an antibody, an antibody having a variable region of a humanized PM1 antibody can be mentioned. The variable region of the humanized PM1 antibody may be subjected to the above-described amino acid substitution, or may be substituted, deleted, added and / or inserted with other amino acids. Specific examples of substitution include the modifications (a) to (y) that improve the affinity described above, the modifications (i) to (viii) that reduce the isoelectric point, and the following (α) to (ζ) Examples include, but are not limited to, modifications that improve stability and modifications that reduce immunogenicity.
As one embodiment of such an antibody, a heavy chain variable region having the amino acid sequence described in SEQ ID NO: 113 (PF_1 + M14ΔGK) and a light chain variable region having the amino acid sequence described in SEQ ID NO: 23 (PF1_L) (The light chain constant region may be kappa, lamda, or a variant thereof), but is not limited thereto.
In addition, the antibody constant region described above and / or the antibody molecule containing the antibody constant region described above can bind an antibody-like binding molecule (scaffold molecule), a bioactive peptide, a binding peptide, or the like as an Fc fusion molecule.

  In addition to the methods described in the Examples, the antibody of the present invention can also be obtained, for example, as follows. In one embodiment of obtaining the antibody of the present invention, first, in at least one region selected from the group consisting of CDR region, FR region, and constant region of anti-IL-6 receptor antibody known to those skilled in the art, 1 Alternatively, a plurality of amino acid residues are substituted with other amino acids of interest. There are no limitations on the method for obtaining anti-IL-6 receptor antibodies known to those skilled in the art. Examples of a method for substituting one or more amino acid residues with other amino acids of interest in at least one region selected from the group consisting of CDR region, FR region, and constant region include, for example, site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M.  (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis.  Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol.  100, 468-500, Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction.  Nucleic Acids Res.  12, 9441-9456, Kramer W, and Fritz HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods.  Enzymol.  154, 350-367, Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A.  82, 488-492). Using this method, the desired amino acid of the antibody can be substituted with another amino acid of interest. As a method for substitution with other amino acids, framework shuffling (Mol Immunol.  By using library technology such as 2007 Apr; 44 (11): 3049-60) and CDR repair (US2006 / 0122377), amino acid substitutions can be made to an appropriate framework and CDR.

  As another embodiment for obtaining an antibody, first, an antibody that binds to IL-6 receptor is obtained by a method well known to those skilled in the art. If the obtained antibody is a non-human animal antibody, it can be humanized. Next, whether or not the obtained antibody has neutralizing activity is determined by a method well known to those skilled in the art. The binding activity or neutralizing activity of the antibody can be measured by, for example, the method described in Examples, but is not limited to this method. Next, one or more amino acid residues in at least one region selected from the group consisting of the CDR region, FR region, and constant region of the antibody are substituted with other amino acids of interest.

More specifically, the present invention includes the following steps (a) and (b), with enhanced neutralizing activity, enhanced binding activity, improved stability, or reduced immunogenicity: The present invention relates to a method for producing the prepared antibody.
(A) DNA encoding an H chain in which one or more amino acid residues in at least one region selected from the group consisting of CDR region, FR region, and constant region are substituted with other amino acids of interest; and A step (b) of expressing DNA encoding an L chain in which one or more amino acid residues in at least one region selected from the group consisting of CDR region and FR region are substituted with other amino acids of interest (step (b)) The step of recovering the expression product of a)

  In the production method of the present invention, first, DNA encoding the H chain of a mutant of an anti-IL-6 receptor antibody, wherein at least one region selected from the group consisting of a CDR region, an FR region, and a constant region DNA encoding an H chain in which one or more amino acid residues therein are substituted with other amino acids of interest, and DNA encoding the L chain of an anti-IL-6 receptor antibody, comprising a CDR region and an FR region DNA encoding an L chain in which at least one amino acid residue in at least one region selected from the group is substituted with another amino acid of interest is expressed. DNA encoding an H chain in which at least one amino acid residue in at least one region selected from the group consisting of CDR region, FR region, and constant region is substituted with another amino acid of interest is, for example, wild A CDR region, FR region, or constant region portion of DNA encoding the H chain of the type is obtained, and a specific amino acid in at least one region selected from the group consisting of the CDR region, FR region, and constant region It can be obtained by introducing appropriate substitutions so that the encoded codon encodes other desired amino acids. In addition, DNA encoding an L chain in which one or more amino acid residues in at least one region selected from the group consisting of CDR region and FR region are substituted with other amino acids of interest, for example, wild type Obtain the CDR region and / or FR region of DNA encoding the L chain, and introduce appropriate substitutions so that the codon encoding a specific amino acid in the CDR region and FR region encodes the other amino acid of interest. Can be obtained by

In addition, a protein in which one or more amino acid residues in at least one region selected from the group consisting of a CDR region, an FR region, and a constant region of a wild-type H chain are substituted with other desired amino acids in advance. By designing the DNA to be encoded and chemically synthesizing the DNA, one or more amino acid residues in at least one region selected from the group consisting of a CDR region, an FR region, and a constant region can be obtained. It is also possible to obtain DNA encoding an H chain substituted with another amino acid. For the L chain, a DNA encoding a protein in which one or more amino acid residues in the CDR region and / or FR region of the wild-type L chain are replaced with other amino acids of interest is designed, and the DNA is chemically It is also possible to obtain DNA encoding an L chain in which one or more amino acid residues in the CDR region and / or FR region are substituted with other amino acids of interest.
The types of amino acid substitutions include, but are not limited to, the substitutions described herein.

  Further, in at least one region selected from the group consisting of a CDR region, an FR region, and a constant region, a DNA encoding an H chain in which one or more amino acid residues are substituted with another amino acid of interest, It can be manufactured by dividing into partial DNA. Examples of the combination of partial DNAs include DNA encoding a variable region and DNA encoding a constant region, or DNA encoding a Fab region and DNA encoding an Fc region, but are not limited to these combinations. is not. Similarly, DNA encoding the L chain can also be produced by dividing it into partial DNAs.

  Examples of the method for expressing the DNA include the following methods. For example, a DNA encoding an H chain variable region is incorporated into an expression vector together with DNA encoding an H chain constant region to construct an H chain expression vector. Similarly, a DNA encoding an L chain variable region is incorporated into an expression vector together with DNA encoding an L chain constant region to construct an L chain expression vector. These H chain and L chain genes can also be incorporated into a single vector. Examples of expression vectors include, but are not limited to, SV40 virus based vectors, EB virus based vectors, BPV (papilloma virus) based vectors, and the like.

  A host cell is cotransformed with the antibody expression vector prepared by the above method. As host cells, in addition to the above-mentioned cells such as CHO cells (Chinese hamster ovary), microorganisms such as Escherichia coli, yeast and Bacillus subtilis, and animals and plants are used (Nature Biotechnology 25, 563-565 (2007), Nature Biotechnology 16). , 773-777 (1998), Biochemical and Biophysical Research Communications 255, 444-450 (1999), Nature Biotechnology 23, 1159-1169 (2005), Journal of Virology 75, 2803-2809 (2001), Biochemical and Biophysical Research Communications 308, 94-100 (2003)). For transformation, the lipofectin method (RWMalone et al., Proc. Natl. Acad. Sci. USA 86, 6077 (1989), PLFelgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 ( 1987), electroporation method, calcium phosphate method (FL Graham & AJvan der Eb, Virology 52, 456-467 (1973)), DEAE-Dextran method and the like are preferably used.

In the production of the antibody, the expression product obtained in step (a) is then recovered. The expression product can be collected, for example, by culturing the transformant and then separating it from the cells of the transformant or the culture solution. For antibody separation and purification, methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, 1q, FcRn, protein A, protein G column, affinity chromatography, ion exchange chromatography, and gel filtration chromatography are used. It can carry out in combination as appropriate.
In addition, the constant region of the present invention can be appropriately performed by those skilled in the art according to the antibody obtaining method.

Furthermore, the present invention enhances the binding activity or neutralizing activity of the anti-IL-6 receptor antibody to the IL-6 receptor, comprising at least one step selected from the group consisting of the following (A) to (X): Regarding the method.
(A) A step of substituting the first Ser with another amino acid in the amino acid sequence shown in SEQ ID NO: 1 (HCDR1)
(B) A step of substituting the 5th Trp with another amino acid in the amino acid sequence (HCDR1) described in SEQ ID NO: 1.
(C) A step of substituting the first Tyr with another amino acid in the amino acid sequence (HCDR2) described in SEQ ID NO: 2
(D) A step of substituting the 8th Thr with another amino acid in the amino acid sequence (HCDR2) described in SEQ ID NO: 2
(E) Substituting the 9th Thr with another amino acid in the amino acid sequence (HCDR2) described in SEQ ID NO: 2
(F) A step of substituting the first Ser with another amino acid in the amino acid sequence (HCDR3) described in SEQ ID NO: 3
(G) A step of substituting the second Leu with another amino acid in the amino acid sequence (HCDR3) described in SEQ ID NO: 3
(H) A step of substituting the 5th Thr with another amino acid in the amino acid sequence (HCDR3) described in SEQ ID NO: 3
(I) A step of substituting the 7th Ala with another amino acid in the amino acid sequence (HCDR3) described in SEQ ID NO: 3
(J) A step of substituting the 8th Met with another amino acid in the amino acid sequence shown in SEQ ID NO: 3 (HCDR3)
(K) A step of substituting the first Ser and the fifth Thr with other amino acids in the amino acid sequence shown in SEQ ID NO: 3 (HCDR3)
(L) A step of substituting the second Leu, the seventh Ala and the eighth Met with other amino acids in the amino acid sequence (HCDR3) described in SEQ ID NO: 3
(M) A step of substituting the first Arg with another amino acid in the amino acid sequence shown in SEQ ID NO: 4 (LCDR1)
(N) A step of substituting the fourth Gln with another amino acid in the amino acid sequence shown in SEQ ID NO: 4 (LCDR1)
(O) A step of substituting the 9th Tyr with another amino acid in the amino acid sequence shown in SEQ ID NO: 4 (LCDR1)
(P) A step of substituting the 11th Asn with another amino acid in the amino acid sequence shown in SEQ ID NO: 4 (LCDR1)
(Q) A step of substituting the second Thr with another amino acid in the amino acid sequence shown in SEQ ID NO: 5 (LCDR2)
(R) A step of substituting the first Gln with another amino acid in the amino acid sequence shown in SEQ ID NO: 6 (LCDR3)
(S) A step of substituting the third Gly with another amino acid in the amino acid sequence shown in SEQ ID NO: 6 (LCDR3)
(T) the step of substituting the 9th Tyr with another amino acid in the amino acid sequence (LCDR1) described in SEQ ID NO: 4 and the 3rd Gly in the amino acid sequence (LCDR3) described in SEQ ID NO: 6 Step to replace
(U) A step of substituting the 5th Thr with another amino acid in the amino acid sequence shown in SEQ ID NO: 6 (LCDR3)
(V) A step of substituting the first Gln and the fifth Thr with other amino acids in the amino acid sequence shown in SEQ ID NO: 6 (LCDR3)
(W) Substituting the 9th Thr with another amino acid in the amino acid sequence (HCDR2) described in SEQ ID NO: 2 and the 1st Ser and 5th in the amino acid sequence (HCDR3) described in SEQ ID NO: 3 Substitution of Thr with another amino acid
(X) The process described in (V) and (W).

In the above (A), the amino acid after substitution is not particularly limited as long as the affinity is improved, but to Trp, Thr, or Asp, Asn, Arg, Val, Phe, Ala, Gln, Tyr, Leu, His, Glu or Cys Is preferred.
In (B) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Ile or Val is preferred.
In (C) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Phe is preferred.
In (D) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Arg is preferred.
In (E) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Ser or Asn is preferred.
In (F) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Ile, Val, Thr or Leu is preferred.
In (G) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Thr is preferred.
In (H) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Ala, Ile or Ser is preferred. Other preferred substitutions include substitution of the fifth Thr to Ser.
In (I) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Ser or Val is preferred.
In (J) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Leu is preferred.
In (K) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but it is preferable to substitute the first Ser with Leu and the fifth Thr with Ala. As other preferred substitutions, the substitution of the first Ser to Val and the substitution of the fifth Thr to Ala, the substitution of the first Ser to Ile and the substitution of the fifth Thr to Ala, the first Substitution of Ser to Thr and substitution of 5th Thr to Ala, substitution of 1st Ser to Val and substitution of 5th Thr to Ile, substitution of 1st Ser to Ile and 5th Substitution of Thr to Ile, substitution of 1st Ser to Thr and substitution of 5th Thr to Ile, or substitution of 1st Ser to Leu and substitution of 5th Thr to Ile be able to.
In (L) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but it is preferable to substitute the second Leu with Thr, the seventh Ala with Val, and the eighth Met with Leu.
In (M) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Phe is preferred.
In (N) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Arg or Thr is preferred.
In (O) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Phe is preferred.
In (P) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Ser is preferred.
In (Q) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Gly is preferred.
In (R) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Gly, Asn or Ser is preferred.
In (S) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Ser is preferred.
In (T) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but Tyr in the amino acid sequence (LCDR1) described in SEQ ID NO: 4 is preferably substituted with Phe, SEQ ID NO: Gly in the amino acid sequence described in 6 (LCDR3) is preferably substituted with Ser.
In (U) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Arg or Ser is preferred.
In (V) above, the amino acid after substitution is not particularly limited as long as the affinity is improved, but it is preferable to substitute the first Gln with Gly and the fifth Thr with Ser. Other preferred substitutions include the first substitution of Gln with Gly and the fifth substitution of Thr with Arg.
In the above (W), the 9th Thr of the amino acid sequence (HCDR2) described in SEQ ID NO: 2 is preferably substituted with Asn. As preferred combinations of amino acids after substitution of the first Ser and the fifth Thr of the amino acid sequence (HCDR3) shown in SEQ ID NO: 3, Leu and Ala, Val and Ala, Ile and Ala, Thr and Ala, Mention may be made of Val and Ile, Ile and Ile, Thr and Ile, or Leu and Ile.

  In the steps (A) to (X) described above, the method for substituting amino acids is not particularly limited. For example, the site-directed mutagenesis method or the method described in the examples can be used. When amino acid substitution is performed on the heavy chain variable region, the amino acid sequence of the heavy chain variable region that is the group before substitution is preferably the amino acid sequence of the heavy chain variable region of humanized PM-1. When is performed on the light chain variable region, the amino acid sequence of the light chain variable region that is the base before substitution is preferably the amino acid sequence of the light chain variable region of humanized PM-1. The amino acid substitution described in the above steps (A) to (X) is preferably performed on the humanized PM-1 antibody.

  The method for enhancing the binding activity or neutralizing activity of the anti-IL-6 receptor antibody of the present invention only needs to include at least the steps described in any of (A) to (X) above. That is, the method of the present invention may include two or more steps among the steps described in any of the above (A) to (X). Furthermore, as long as the process described in any of the above (A) to (X) is included, other processes (for example, substitution, deletion, addition and / or substitution of amino acids other than the above (A) to (X)) Or insertion etc.). Further, for example, substitution, deletion, addition and / or insertion of FR amino acid sequence may be performed, and substitution, deletion, addition and / or insertion of constant region amino acid sequence may be performed. Also good. The amino acid substitution described above is preferably performed on a humanized PM-1 antibody.

<Method of reducing the immunogenicity risk of anti-IL-6 receptor antibody>
The present invention also relates to a method for reducing the immunogenicity of an anti-IL-6 receptor antibody, comprising a step of substituting the second Thr with Gly in the amino acid sequence shown in SEQ ID NO: 5 (LCDR2). As long as the method for reducing the immunogenicity of the anti-IL-6 receptor antibody of the present invention includes the step of substituting the second Thr with Gly in the amino acid sequence shown in SEQ ID NO: 5 (LCDR2), other amino acid substitutions May be included. The method of amino acid substitution is not particularly limited, and for example, it can be carried out by the above-mentioned site-directed mutagenesis method or the method described in the examples.
The amino acid substitution described above is preferably performed on the humanized PM-1 antibody or a modified (substitution, deletion, insertion) form thereof.

<Method of reducing isoelectric point of anti-IL-6 receptor antibody>
The present invention also relates to a method for reducing the isoelectric point of an anti-IL-6 receptor antibody, comprising at least one step selected from the group consisting of the following (i) to (xv).
(i) A step of substituting the 16th Gln with another amino acid in the amino acid sequence (HFR1) described in SEQ ID NO: 7
(ii) A step of substituting the 8th Arg with another amino acid in the amino acid sequence (HFR2) described in SEQ ID NO: 8
(iii) A step of substituting the 16th Arg with another amino acid in the amino acid sequence shown in SEQ ID NO: 9 (HFR3)
(iv) A step of substituting the third Gln with another amino acid in the amino acid sequence (HFR4) described in SEQ ID NO: 10
(v) A step of substituting the 18th Arg with another amino acid in the amino acid sequence (LFR1) described in SEQ ID NO: 11
(vi) a step of substituting the 11th Lys with another amino acid in the amino acid sequence (LFR2) described in SEQ ID NO: 12
(vii) A step of substituting the 23rd Gln with another amino acid in the amino acid sequence shown in SEQ ID NO: 13 (LFR3)
(viii) A step of substituting the 10th Lys with another amino acid in the amino acid sequence shown in SEQ ID NO: 14 (LFR4)
(ix) A step of substituting the first Ser with another amino acid in the amino acid sequence (HCDR1) described in SEQ ID NO: 1
(x) substituting the first Arg with another amino acid in the amino acid sequence (LCDR1) described in SEQ ID NO: 4
(xi) A step of substituting the fourth Arg with another amino acid in the amino acid sequence shown in SEQ ID NO: 5 (LCDR2)
(xii) a step of substituting the 13th Arg with another amino acid in the amino acid sequence (HFR1) described in SEQ ID NO: 7
(xiii) A step of substituting the 15th Lys and / or 16th Ser in the amino acid sequence (HFR1) described in SEQ ID NO: 2 or the amino acid sequence described in SEQ ID NO: 100 with another amino acid.
(xiv) A step of substituting the fourth Gln with another amino acid in the amino acid sequence (LCDR1) described in SEQ ID NO: 4 or the amino acid sequence described in SEQ ID NO: 101
(xv) A step of substituting the sixth His in the amino acid sequence shown in SEQ ID NO: 5 (LCDR2) or the amino acid sequence shown in SEQ ID NO: 103 with another amino acid

In (i) above, the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Glu is preferred.
In (ii) above, the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Glu is preferred.
In the above (iii), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution to Lys is preferable.
In (iv) above, the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution to Glu is preferred.
In the above (v), the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Ser is preferred.
In (vi) above, the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Glu is preferred.
In (vii) above, the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution to Glu is preferred.
In the above (viii), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution to Glu is preferred.
In (ix) above, the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Asp is preferred.
In (x) above, the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Gln is preferred.
In (xi) above, the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Glu is preferred.
In (xii) above, the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Lys is preferred.
In the above (xiii), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution of 15th Lys to Gln and 16th Ser to Asp is preferable.
In (xiv) above, the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Glu is preferred.
In (xv) above, the amino acid after substitution is not particularly limited as long as the isoelectric point decreases, but substitution to Glu is preferred.

In the steps (i) to (xv) described above, the amino acid substitution method is not particularly limited. For example, the site-directed mutagenesis method or the method described in the examples can be used. When amino acid substitution is performed on the heavy chain variable region, the amino acid sequence of the heavy chain variable region that is the group before substitution is preferably the amino acid sequence of the heavy chain variable region of humanized PM-1. When is performed on the light chain variable region, the amino acid sequence of the light chain variable region, which is the group before substitution, is preferably the amino acid sequence of the light chain variable region of humanized PM-1. In addition, the amino acid substitution described in the above steps (i) to (xv) is preferably performed on the humanized PM-1 antibody.
The method for lowering the isoelectric point of the anti-IL-6 receptor antibody of the present invention only needs to include at least the steps described in any of (i) to (xv) above. That is, the method of the present invention may include two or more steps among the steps described in (i) to (xv) above. Furthermore, as long as the steps described in any of the above (i) to (xv) are included, other steps (for example, substitution, deletion, addition and / or amino acid other than the above (i) to (xv)) Or insertion etc.). Further, for example, substitution, deletion, addition and / or insertion of the amino acid sequence of the constant region may be performed.

<Method for improving anti-IL-6 receptor antibody stability>
The present invention also relates to a method for increasing the stability of an anti-IL-6 receptor antibody, comprising at least one step selected from the group consisting of the following (α) to (ζ).
(α) A step of substituting the fourth Met with another amino acid in the amino acid sequence (HFR3) described in SEQ ID NO: 9.
(β) A step of substituting the 5th Leu with another amino acid in the amino acid sequence (HFR3) described in SEQ ID NO: 9.
(γ) A step of substituting the 9th Thr with another amino acid in the amino acid sequence (HCDR2) described in SEQ ID NO: 2.
(δ) A step of substituting the fifth Thr with another amino acid in the amino acid sequence (LCDR3) described in SEQ ID NO: 6.
(ε) A step of substituting the 16th Ser in the amino acid sequence (HCDR2) described in SEQ ID NO: 2 with another amino acid.
(ζ) A step of substituting the 5th Ser with another amino acid in the amino acid sequence (FR4) described in SEQ ID NO: 10.
In (α) above, the amino acid after substitution is not particularly limited as long as the stability increases, but substitution to Ile is preferred.
In (β) above, the amino acid after substitution is not particularly limited as long as the stability increases, but substitution to Ser is preferred.
In (γ) above, the amino acid after substitution is not particularly limited as long as the stability increases, but substitution to Asn is preferred.
In (δ) above, the amino acid after substitution is not particularly limited as long as the stability increases, but substitution to Ser is preferred.
In (ε) above, the amino acid after substitution is not particularly limited as long as the stability increases, but substitution to Gly is preferred.
In the above (ζ), the amino acid after substitution is not particularly limited as long as the stability increases, but substitution to Ile is preferred.

In the steps (α) to (ζ) described above, the amino acid substitution method is not particularly limited. For example, it can be performed by the above-described site-specific mutagenesis method or the method described in Examples. When amino acid substitution is performed on the heavy chain variable region, the amino acid sequence of the heavy chain variable region that is the group before substitution is preferably the amino acid sequence of the heavy chain variable region of humanized PM-1. When is performed on the light chain variable region, the amino acid sequence of the light chain variable region, which is the group before substitution, is preferably the amino acid sequence of the light chain variable region of humanized PM-1. In addition, the above-mentioned amino acid substitutions (α) to (ζ) are preferably performed on the humanized PM-1 antibody.
The method for increasing the stability of the anti-IL-6 receptor antibody of the present invention only needs to include at least the steps described in any of the above (α) to (ζ). That is, the method of the present invention may include two or more steps among the steps described in the above (α) to (ζ). Furthermore, as long as the process described in any one of the above (α) to (ζ) is included, other processes (for example, substitution, deletion, addition and / or substitution of amino acids other than the above (α) to (ζ)) Or insertion etc.). Further, for example, substitution, deletion, addition and / or insertion of the amino acid sequence of the constant region may be performed.

<Method for reducing immunogenicity of anti-IL-6 receptor antibody>
Furthermore, the present invention relates to the amino acid sequence (HFR1) set forth in SEQ ID NO: 7, wherein the 13th Arg is Lys, the 16th Gln is Glu, the 23rd Thr is Ala, and / or the 30th Thr is Ser. And a method for reducing the immunogenicity of an anti-IL-6 receptor antibody, particularly a humanized PM-1 antibody. The method for reducing the immunogenicity of the anti-IL-6 receptor antibody of the present invention includes other amino acid substitutions as long as it includes the step of substituting the 30 th Thr with Ser in the amino acid sequence (HFR1) described in SEQ ID NO: 7. May be included.
Furthermore, the present invention includes a step of substituting Val at position 27 in the amino acid sequence (HFR3) set forth in SEQ ID NO: 90 with Val to reduce the immunogenicity of an anti-IL-6 receptor antibody, particularly a humanized PM-1 antibody. It relates to the method of making it. As long as the method for reducing the immunogenicity of the anti-IL-6 receptor antibody of the present invention includes the step of substituting Val at the 27th Ala in the amino acid sequence shown in SEQ ID NO: 90 (HFR3), other amino acid substitutions May be included.
The method of amino acid substitution is not particularly limited, and for example, it can be carried out by the above-mentioned site-directed mutagenesis method or the method described in the examples.
Furthermore, the present invention relates to anti-IL-6 receptor antibody, particularly humanized PM-1 antibody, H53 / L28, or FR3 of PF1 antibody by changing FR3 having the amino acid sequence of SEQ ID NO: 128 or SEQ ID NO: 129. It relates to a method for reducing immunogenicity.

<Method for improving the stability of antibodies under acidic conditions>
The present invention also relates to the stability of an antibody under acidic conditions, comprising a step of substituting Met at position 276 (EU numbering 397) with another amino acid in the amino acid sequence (IgG2) represented by SEQ ID NO: 20. It relates to a method of improving. The method for improving the stability of the antibody of the present invention under acidic conditions comprises a step of substituting Met at the 276th position (397th position of EU numbering) with another amino acid in the amino acid sequence (IgG2) represented by SEQ ID NO: 20. Other amino acid substitutions may be included as long as they are included. The amino acid after substitution is not particularly limited, but substitution to Val is preferable. The method of amino acid substitution is not particularly limited, and for example, it can be performed by the above-described site-directed mutagenesis method or the method described in the examples.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.

<Method for improving heterogeneity derived from the hinge region of IgG2 constant region>
In the amino acid sequence (IgG2) shown in SEQ ID NO: 20, the present invention comprises a step of substituting Cys at position 14 (EU numbering 131) with another amino acid, Arg at position 16 (EU numbering 133). The present invention relates to a method for improving heterogeneity of an antibody, comprising a step of substituting with another amino acid and / or substituting Cys at position 102 (219 of EU numbering) with another amino acid. The amino acid after substitution is not particularly limited, but it is preferred that the 14th Cys is substituted with Ser, the 16th Arg is substituted with Lys, and the 102nd Cys is substituted with Ser. The method for improving the heterogeneity of the antibody of the present invention comprises the step of substituting the 14th (EU numbering 131) Cys in the amino acid sequence (IgG2) set forth in SEQ ID NO: 20, and the 16th (EU numbering 133). As long as it includes the step of substituting Arg of No.) and / or the substitution of Cys at No. 102 (219 of EU numbering). The method of amino acid substitution is not particularly limited, and for example, it can be carried out by the above-mentioned site-directed mutagenesis method or the method described in the examples. As the amino acid to be substituted, all three amino acids described above may be substituted, or 1 or 2 (for example, 14th and 102nd amino acids) may be substituted.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.

<Method for reducing heterogeneity derived from C-terminal amino acid deletion of IgG2 constant region>
The present invention also includes a step of deleting the 325th (EU numbering 446th) Gly and the 326th (EU numbering 447th) Lys in the IgG2 constant region having the amino acid sequence of SEQ ID NO: 20. And to a method for improving the heterogeneity of antibodies. The method for improving the heterogeneity of the antibody of the present invention is as follows. In the IgG2 constant region having the amino acid sequence shown in SEQ ID NO: 20, the 325th (EU numbering 446th) Gly and the 326th (EU numbering 447th) Other amino acid substitutions may be included as long as the method includes a step of deleting Lys. The method of amino acid substitution is not particularly limited, and for example, it can be carried out by the above-mentioned site-directed mutagenesis method or the method described in the examples.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.

<Method for reducing binding to FcγR while maintaining the human sequence of IgG2 constant region>
In the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, the present invention includes a step of substituting 209th (EU330) Ala with Ser, a step of substituting 210th (EU331) Pro with Ser, and The present invention relates to a method for reducing the binding of an antibody to FcγR, comprising a step of substituting 218th (EU339) Thr with Ala. The method of reducing the binding of the antibody of the present invention to FcγR comprises the step of substituting Ser at the 209th (EU330) Ala in the IgG2 constant region having the amino acid sequence of SEQ ID NO: 20, 210th (EU331) As long as it includes the step of substituting Pro with Ser and the step of substituting 218 th (EU339) Thr with Ala, other amino acid substitutions may be included. The method of amino acid substitution is not particularly limited, and for example, it can be carried out by the above-mentioned site-directed mutagenesis method or the method described in the examples.

<Method of improving plasma retention by replacing amino acid in IgG2 constant region>
In the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, the present invention provides other amino acids such as His at position 147 (EU268), Arg at position 234 (EU355) and / or Gln at position 298 (EU419). The present invention relates to a method for improving the retention of antibodies in plasma, comprising the step of substituting The method for improving plasma retention of the present invention may include other amino acid substitutions as long as it includes the steps described above. The amino acid after substitution is not particularly limited, but it is preferable that His at position 147 (EU268) is replaced with Gln, Arg at position 234 (EU355) is replaced with Gln, and Gln at position 298 (EU419) is replaced with Glu.
The present invention also includes the step of substituting the 313th (EU434) Asn with another amino acid in the IgG2 constant region having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 151 (M58). The present invention relates to a method for improving performance. The amino acid after substitution is not particularly limited, but substitution to Ala is preferable. The method for improving plasma retention of the present invention may include other amino acid substitutions as long as it includes the steps described above.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.

The present invention also relates to a method for reducing heterogeneity derived from the hinge portion of IgG2, comprising the steps described below, in an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, stable under acidic conditions The present invention relates to a method for improving sex, a method for reducing heterogeneity derived from the C-terminus, and / or a method for reducing antibody binding to FcγR (M14ΔGK).
(A) the step of substituting Ser at 209 (EU numbering 330) of SEQ ID NO: 20 with Ser;
(B) a step of substituting Pro at 210th position (EU numbering 331) of SEQ ID NO: 20 with Ser;
(C) replacing 218 th (EU numbering 339) Thr of SEQ ID NO: 20 with Ala;
(D) a step of substituting Val at the 276th (EU numbering 397) of SEQ ID NO: 20;
(E) the step of substituting the 14th (EU numbering 131) Cys of SEQ ID NO: 20 with Ser;
(F) a step of replacing Arg at the 16th position (EU numbering 133) of SEQ ID NO: 20 with Lys;
(G) a step of substituting Cy at the 102nd position (EU numbering 219) of SEQ ID NO: 20 with Ser;
(H) a step of substituting Glu at the 20th (EU numbering 137) of SEQ ID NO: 20 with Gly;
(I) replacing 21st (EU numbering 138) Ser of SEQ ID NO: 20 with Gly;
And (j) deleting the 325th Gly and the 326th Lys (EU numbering 446 and 447) of SEQ ID NO: 20.
The method of the present invention may include other amino acid substitutions, deletions, and other steps as long as the above steps are included. The method of amino acid substitution or deletion is not particularly limited, and can be performed by, for example, the above-described site-directed mutagenesis method or the method described in the examples.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.

The present invention also relates to a method for reducing heterogeneity derived from the hinge portion of IgG2, comprising the steps described below, in an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, stable under acidic conditions The present invention relates to a method for improving sex and / or a method for reducing heterogeneity derived from the C-terminus (M31ΔGK).
(A) replacing Met at position 276 (EU numbering 397) of SEQ ID NO: 20 with Val;
(B) a step of substituting the 14th (EU numbering 131) Cys of SEQ ID NO: 20 with Ser;
(C) replacing Arg at the 16th position (EU numbering 133) of SEQ ID NO: 20 with Lys;
(D) a step of substituting Cy at position 102 (EU numbering 219) of SEQ ID NO: 20 with Ser;
(E) a step of replacing Glu at the 20th (EU numbering 137) of SEQ ID NO: 20 with Gly;
(F) a step of substituting the 21st (EU numbering 138) Ser of SEQ ID NO: 20 with Gly;
And (g) a step of deleting 325th Gly and 326th Lys (EU numbering 446 and 447) of SEQ ID NO: 20.

The present invention also relates to a method for reducing heterogeneity derived from the hinge portion of IgG2, including the steps described below, in an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, and to improve plasma retention. The method and / or the method for reducing heterogeneity derived from the C-terminus (M58).
(A) replacing the 14th (EU numbering 131) Cys of SEQ ID NO: 20 with Ser;
(B) a step of replacing Arg at the 16th position (EU numbering position 133) of SEQ ID NO: 20 with Lys;
(C) the step of substituting the 102nd (EU numbering 219th) Cys of SEQ ID NO: 20 with Ser;
(D) a step of substituting Glu for the 20th (EU numbering 137th) Glu of SEQ ID NO: 20;
(E) replacing the 21st (EU numbering 138th) Ser of SEQ ID NO: 20 with Gly;
(F) replacing 147th (EU numbering 268th) His of SEQ ID NO: 20 with Gln;
(G) a step of substituting Arn at 234th (EU numbering 355th) of SEQ ID NO: 20 with Gln;
(H) a step of substituting Glu for Gln at the 298th (EU numbering 419th) position of SEQ ID NO: 20;
(I) A step of deleting 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys of SEQ ID NO: 20.

The present invention also relates to a method for reducing heterogeneity derived from the hinge portion of IgG2, including the steps described below, in an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, and to improve plasma retention. The method and / or the method for reducing heterogeneity derived from the C-terminus (M73).
(A) replacing the 14th (EU numbering 131) Cys of SEQ ID NO: 20 with Ser;
(B) a step of replacing Arg at the 16th position (EU numbering position 133) of SEQ ID NO: 20 with Lys;
(C) the step of substituting the 102nd (EU numbering 219th) Cys of SEQ ID NO: 20 with Ser;
(D) a step of substituting Glu for the 20th (EU numbering 137th) Glu of SEQ ID NO: 20;
(E) replacing the 21st (EU numbering 138th) Ser of SEQ ID NO: 20 with Gly;
(F) replacing 147th (EU numbering 268th) His of SEQ ID NO: 20 with Gln;
(G) a step of substituting Arn at 234th (EU numbering 355th) of SEQ ID NO: 20 with Gln;
(H) a step of substituting Glu for Gln at the 298th (EU numbering 419th) position of SEQ ID NO: 20;
(I) the step of substituting 313th (EU numbering 434th) Asn of SEQ ID NO: 20 with Ala;
(J) A step of deleting 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys of SEQ ID NO: 20.

The present invention also relates to a method for reducing heterogeneity derived from the hinge part of IgG2, comprising the steps described below, in the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, heterozygous derived from the C-terminus. It relates to a method for reducing the gender and / or a method for reducing the binding of an antibody to FcγR (M86ΔGK).
(A) the step of substituting 209th (EU numbering 330) Ala of SEQ ID NO: 20 with another amino acid;
(B) substituting Pro of SEQ ID NO: 20 at position 210 (EU numbering 331) with another amino acid;
(C) substituting Thr at 218th (EU numbering 339) of SEQ ID NO: 20 with another amino acid;
(D) a step of substituting Cys at the 14th position (EU numbering 131) of SEQ ID NO: 20 with another amino acid;
(E) substituting Arg at the 16th position (EU numbering 133) of SEQ ID NO: 20 with another amino acid;
(F) a step of substituting Cys at position 102 (EU numbering 219) of SEQ ID NO: 20 with another amino acid;
(G) the step of substituting the 20th (EU numbering 137) Glu of SEQ ID NO: 20 with another amino acid;
(H) substituting Ser at the 21st position (EU numbering 138) of SEQ ID NO: 20 with another amino acid;
And (i) a step of deleting 325th Gly and 326th Lys (EU numbering 446 and 447) of SEQ ID NO: 20.
The amino acid after substitution is not particularly limited, but the 209th (EU numbering 330) Ala is Ser, the 210th (EU numbering 331) Pro is Ser, the 218th (EU numbering 339) Thr is Ala, the 14th (EU Numbering 131) Cys is Ser, 16th (EU numbering 133) Arg is Lys, 102th (EU numbering 219) Cys is Ser, 20th (EU numbering 137) Glu is Gly, 21st (EU numbering 138) ) Ser is preferably replaced with Gly.

Furthermore, the present invention relates to a method for reducing heterogeneity derived from the hinge portion of IgG2 and / or derived from the C-terminus, comprising the steps described below in the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20. The present invention relates to a method for reducing heterogeneity (M40ΔGK).
(A) a step of substituting Cys at position 14 (EU numbering 131) of SEQ ID NO: 20 with another amino acid;
(B) a step of substituting Arg at the 16th position (EU numbering 133) of SEQ ID NO: 20 with another amino acid;
(C) a step of substituting Cys at position 102 (EU numbering 219) of SEQ ID NO: 20 with another amino acid;
(D) a step of substituting the 20th (EU numbering 137) Glu of SEQ ID NO: 20 with another amino acid;
(E) substituting the 21st (EU numbering 138) Ser of SEQ ID NO: 20 with another amino acid;
And (f) a step of deleting 325th Gly and 326th Lys (EU numbering 446 and 447) of SEQ ID NO: 20.
The amino acid after substitution is not particularly limited, but the 14th (EU numbering 131) Cys is Ser, the 16th (EU numbering 133) Arg is Lys, the 102nd (EU numbering 219) Cys is Ser, and the 20th (EU It is preferable to substitute Gly for Glu in the numbering 137) and Gly for the 21st (EU numbering 138) Ser.

The method of the present invention may include other amino acid substitutions, deletions, and other steps as long as the above steps are included. The method of amino acid substitution or deletion is not particularly limited, and can be performed by, for example, the above-described site-directed mutagenesis method or the method described in the examples.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.

<Method for improving stability of IgG4 constant region under acidic conditions>
In the IgG4 constant region (Mol Immunol. 1993 Jan; 30 (1): 105-8.) Having the amino acid sequence set forth in SEQ ID NO: 21, the present invention also includes Arg at position 289 (EU numbering 409). The present invention relates to a method for improving the stability of an antibody under acidic conditions, which comprises the step of substituting the amino acid. In the method for improving the stability of the antibody of the present invention under acidic conditions, Arg at position 289 (EU numbering 409) is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 21 (human IgG4 constant region). Other amino acid substitutions may be included as long as the steps are included. The amino acid after substitution is not particularly limited, but substitution to Lys is preferable. The method of amino acid substitution is not particularly limited, and for example, it can be carried out by the above-mentioned site-directed mutagenesis method or the method described in the examples.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.

<Method for reducing heterogeneity derived from C-terminal amino acid deletion of IgG4 constant region>
In the IgG4 constant region (Mol Immunol. 1993 Jan; 30 (1): 105-8.) Having the amino acid sequence set forth in SEQ ID NO: 21, the present invention also includes Gly at position 326 (EU numbering 446). The present invention relates to a method for improving the heterogeneity of an antibody, comprising the step of deleting 327th (EU numbering 447) Lys. The method for improving heterogeneity according to the present invention is the 327th (EU numbering) in the IgG4 constant region (Mol Immunol. 1993 Jan; 30 (1): 105-8.) Having the amino acid sequence of SEQ ID NO: 21. Other amino acid substitutions may be included, as long as it includes a step of deleting Ly of 447) and / or Gly of 326 (EU numbering 446). The method of amino acid substitution is not particularly limited, and for example, it can be carried out by the above-mentioned site-directed mutagenesis method or the method described in the examples.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.

The present invention also relates to acidic conditions for IgG4, which comprises the steps described below in the IgG4 constant region (Mol Immunol. 1993 Jan; 30 (1): 105-8.) Having the amino acid sequence set forth in SEQ ID NO: 21. The present invention relates to a method for improving the stability at the bottom, a method for reducing heterogeneity derived from the C-terminus, and a method for reducing the binding of an antibody to FcγR (M11ΔGK).
(A) the step of substituting the 14th (EU numbering 131) Cys of SEQ ID NO: 21 with Ser;
(B) a step of replacing Arg at the 16th position (EU numbering 133) of SEQ ID NO: 21 with Lys;
(C) the step of substituting Glu at the 20th (EU numbering 137) of SEQ ID NO: 21 with Gly;
(D) replacing 21st (EU numbering 138) Ser of SEQ ID NO: 21 with Gly;
(E) substituting Arg of SEQ ID NO: 21 at position 97 (EU numbering 214) with Thr;
(F) a step of substituting Ser at the 100th position (EU numbering 217) of SEQ ID NO: 21 with Arg;
(G) a step of substituting Tyr at the 102nd position (EU numbering 219) of SEQ ID NO: 21 with Ser;
(H) replacing Gly at position 103 (EU numbering 220) of SEQ ID NO: 21 with Cys;
(I) a step of replacing Pro at SEQ ID NO: 21 at position 104 (EU numbering 221) with Val;
(J) a step of replacing the 105th (EU numbering 222) Pro in SEQ ID NO: 21 with Glu;
(K) a step of replacing Glu at the 113th position (EU numbering 233) of SEQ ID NO: 21 with Pro;
(L) a step of replacing Phe at position 114 (EU numbering 234) of SEQ ID NO: 21 with Val;
(M) a step of substituting Lea at 115th (EU numbering 235) of SEQ ID NO: 21 with Ala;
(N) a step of deleting Gly at 116th position (EU numbering 236) of SEQ ID NO: 21;
(O) replacing Arg at position 289 (EU numbering 409) of SEQ ID NO: 21 with Lys; and (p) Gly at position 326 (EU numbering 446) of SEQ ID NO: 21 and position 327 (EU numbering 447). Deficient Lys.
The method of the present invention may include other amino acid substitutions, deletions, and other steps as long as the above steps are included. The method of amino acid substitution or deletion is not particularly limited, and can be performed by, for example, the above-described site-directed mutagenesis method or the method described in the examples.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.

<Method for reducing heterogeneity derived from C-terminal amino acid deletion of IgG1 constant region>
Furthermore, the present invention includes a step of deleting Gly and Gly at position 329 (EU numbering 446) and Lys at position 330 (EU numbering 447) in the IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19. And to a method for improving the heterogeneity of antibodies. In the IgG1 constant region having the amino acid sequence of SEQ ID NO: 19, the method for improving the heterogeneity of the antibody of the present invention is the 330th (EU numbering 447) Lys and 329th (EU numbering 446th). Other amino acid substitutions may be included as long as the method includes a step of deleting Gly. The method of amino acid substitution is not particularly limited, and for example, it can be carried out by the above-mentioned site-directed mutagenesis method or the method described in the examples.

<Method of improving plasma retention by substituting amino acids in IgG1 constant region>
The present invention also relates to a method for improving plasma retention of an antibody, comprising a step of substituting 317th (EU434) Asn with another amino acid in the IgG1 constant region having the amino acid sequence of SEQ ID NO: 19. . The amino acid after substitution is not particularly limited, but substitution to Ala is preferable. The method for improving plasma retention of the present invention may include other amino acid substitutions as long as it includes the steps described above.
The present invention also relates to a method for improving retention in plasma and / or a method for reducing heterogeneity derived from the C-terminus, comprising the steps described below, in an IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19. (M83).
(A) substituting 317th (EU434) Asn of SEQ ID NO: 19 with another Ala;
(B) Deletion of 330th (EU numbering 447th) Lys and 329th (EU numbering 446th) Gly of SEQ ID NO: 19.
The target antibody is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody, or a modified (substitution, deletion, insertion) form thereof.
The above-mentioned constant region of the present invention can be combined with any antibody-derived variable region, but is preferably combined with an antibody-derived variable region against human IL-6 receptor. Examples of the variable region of an antibody against the human IL-6 receptor include the variable region of a humanized PM-1 antibody. The variable region of the humanized PM-1 antibody may be a variable region in which amino acid substitution or the like has not been performed, or may be a variable region in which the above-described amino acid substitution or the like has been performed.

The present invention provides a pharmaceutical composition comprising the antibody of the present invention. The pharmaceutical composition of the present invention is useful in the treatment of diseases associated with IL-6 such as rheumatoid arthritis.
The pharmaceutical composition of the present invention can be formulated by a known method by introducing a pharmaceutically acceptable carrier in addition to the antibody. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension. For example, a pharmacologically acceptable carrier or medium, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to formulate by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredients in these preparations is such that an appropriate volume within the indicated range can be obtained.

Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50 may be used in combination.

  Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. Moreover, you may mix | blend with buffering agents, such as a phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizers, such as benzyl alcohol, phenol, and antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

  Administration is preferably parenteral administration, and specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

  The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

The correspondence between the three-letter code and the one-character code of amino acids used in the present specification is as follows.
Alanine: Ala: A
Arginine: Arg: R
Asparagine: Asn: N
Aspartic acid: Asp: D
Cysteine: Cys: C
Glutamine: Gln: Q
Glutamate: Glu: E
Glycine: Gly: G
Histidine: His: H
Isoleucine: Ile: I
Leucine: Leu: L
Lysine: Lys: K
Methionine: Met: M
Phenylalanine: Phe: F
Proline: Pro: P
Serine: Ser: S
Threonine: Thr: T
Tryptophan: Trp: W
Tyrosine: Tyr: Y
Valine: Val: V

  It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
[Example 1] Improvement of antigen binding ability by CDR modification using affinity maturation technology
Preparation of SR344
Soluble human IL-6R (hereinafter referred to as SR344) consisting of the 1st to 344th amino acid sequences on the N-terminal side reported in J. Biochem. 108, 673-676 (1990) (Yamasaki et al., Science 1988; 241: 825 -828 (GenBank # X12830)) was produced.
From the culture supernatant obtained from SR344-expressing CHO cells, SR344 was purified by three column chromatography: Blue Sepharose 6 FF column chromatography, affinity chromatography using a column with a specific antibody against SR344, and gel filtration column chromatography. did.
The culture supernatant was directly added to a Blue Sepharose 6 FF column (GE Healthcare Bioscience) equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and the non-adsorbed fraction was completely washed away with the same buffer. Thereafter, the column was washed with the same buffer containing 300 mM KCl. Further, the adsorbed protein was eluted with a linear concentration gradient of KSCN from 0 M to 0.5 M in the same buffer in the presence of 300 mM KCl. Fractions eluted with a concentration gradient of KSCN were analyzed by Western Blotting using an antibody specific for SR344, and fractions containing SR344 were collected.
The column on which the specific antibody against SR344 was immobilized was previously equilibrated with TBS (Tris-Buffered-Saline). To this, the SR344 fraction obtained in the first step was concentrated by ultrafiltration with Amicon Ultra-15 (MILLIPORE, molecular weight cut-off 10 kDa), diluted 2 times with TBS and then added. After washing the column with TBS, the adsorbed protein was eluted with 100 mM glycine-hydrochloric acid buffer, pH 2.5. The eluted fraction was returned to neutral pH by adding 1 M Tris, pH 8.1. The obtained fractions were analyzed by SDS-PAGE, and fractions containing SR344 were collected.
The fraction obtained in the second step was concentrated with Amicon Ultra-15 (molecular weight cut-off 10 kDa) and added to a Superdex200 column (GE Healthcare Bioscience) equilibrated with PBS. The fraction eluted as the main peak was used as the final purified product of SR344.

Establishment of BaF3 cell line expressing human gp130
In order to obtain a cell line exhibiting IL-6-dependent growth, a BaF3 cell line expressing human gp130 was established as shown below.
Full-length human gp130 cDNA (Hibi et al., Cell 1990; 63: 1149-1157 (GenBank # NM_002184)) was amplified by PCR to remove the DHFR gene expression site of pCHOI (Hirata et al., FEBS Letter 1994; 356: 244-248) And cloned into an expression vector pCOS2Zeo into which a Zeocin resistance gene expression site was inserted to construct pCOS2Zeo / gp130. Full-length human IL-6R cDNA was amplified by PCR and cloned into pcDNA3.1 (+) (Invitrogen) to construct hIL-6R / pcDNA3.1 (+).
10 μg of pCOS2Zeo / gp130 was mixed with BaF3 cells (0.8 × 10 ⁷ cells) suspended in PBS, and pulsed at a volume of 0.33 kV and 950 μFD using Gene Pulser (Bio-Rad). BaF3 cells transfected by electroporation were cultured overnight in RPMI1640 medium (Invitrogen) containing 0.2 ng / mL mouse interleukin-3 (Peprotech) and 10% Fetal Bovine Serum (hereinafter FBS, HyClone). Selection was performed by adding RPMI1640 medium containing mL of human interleukin-6 (R & D systems), 100 ng / mL human interleukin-6 soluble receptor (R & D systems) and 10% FBS, and a human gp130-expressing BaF3 cell line (hereinafter referred to as BaF3). / gp130) was established. Since this BaF / gp130 grows in the presence of human interleukin-6 (R & D systems) and SR344, it should be used to evaluate the anti-IL-6 receptor antibody growth inhibitory activity (ie, IL-6 receptor neutralizing activity). Is possible.

Construction of CDR-modified library First, scFv of a humanized PM1 antibody (Cancer Res. 1993 Feb 15; 53 (4): 851-6) was performed. VH and VL regions were amplified by PCR, and humanized PM1 HL scFv having a linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 106) between VH and VL was prepared.
By using the prepared humanized PM1 HL scFv DNA as a template, a target library in which one amino acid of each CDR amino acid is X and a library in which only the hot spot sequence in the CDR was randomized were prepared. . For a target library in which one of each CDR amino acid is X, the library part is constructed by PCR reaction using a primer with NNS as the amino acid to be libraryed, and the other part is obtained by normal PCR. Fabricated and assembled by assembly PCR method. At this time, only one CDR was made into a library (see J. Mol. Biol 1996; 256: 77-88). Further, a library in which only the Hot Spot sequence was changed to a random sequence was similarly constructed by PCR using a primer in which all Hot Spot amino acids were NNS. At this time, a library in which only the VH hot spot was converted into a library and a library in which only the VL hot spot was converted into a library were constructed (see Nature Biotechnology 1999 June; 17: 568-572).
Using these libraries, a library for ribosome display was constructed according to J. Immunological Methods 1999; 231: 119-135. In order to perform E. coli cell-free in vitro translation, add SDA sequence (ribosome binding site) and T7 promoter to 5 'side, and ligation gene3 partial sequence on 3' side as linker for ribosome display using Sfi I did.

Acquisition of high affinity scFv by Ribosome display
We learned from Nature Biotechnology 2000 Dec; 18: 1287-1292 and panned by ribosome display. The prepared SR344 was biotinized with NHS-PEO4-Biotin (Pierce) to obtain an antigen. In order to efficiently obtain scFv with high affinity, off-rate selection was performed with reference to JBC 2004; 279 (18): 18870-18877. The amount of biotinylated antigen was 1 nM, the amount of competitor antigen was 1 uM, and the competition time at 4 ^th round was 10 O / N.

scFv insertion into phagemide, antigen binding activity, sequence analysis
The HL scFv was restored by PCR using specific primers with the DNA pool obtained in 4 ^th round as a template. It was digested with Sfi I, similarly inserted into the phagemide vector pELBG lacI vector digested with Sfi I, and transformed into XL1-Blue (stratagene). Using the obtained colonies, antigen binding activity evaluation and HL scFv sequence analysis by phage ELISA were performed. According to J. Mol. Biol 1992; 227: 381-388, a phage-ELISA using a plate coated with SR344 at 1 ug / mL was performed. For clones that showed SR344 binding activity, sequence analysis was performed using specific primers.

IgG was expressed from scFv using IgG vector and IgG expression and purified animal cell expression vector. VH and VL were each amplified by PCR for clones in which mutation sites were concentrated, and inserted into animal cell expression vectors by XhoI / NheI digestion and EcoRI digestion. The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the DNA sequencer ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached instructions.

Expression of antibody converted to IgG Expression of antibody was carried out using the following method. HEK293H strain derived from human fetal kidney cancer cells (Invitrogen) is suspended in DMEM medium (Invitrogen) containing 10% Fetal Bovine Serum (Invitrogen), and the dish (diameter) is used at a cell density of 5-6 x 10 ⁵ cells / mL. 10 mL to each dish of 10 cm, CORNING) After incubating overnight in a CO ₂ incubator (37 ° C, 5% CO ₂ ), the medium is removed by suction and CHO-S-SFM-II (Invitrogen) medium 6.9 mL was added. The prepared plasmid DNA mixture (13.8 μg in total) was mixed with 20.7 μL of 1 μg / mL Polyethylenimine (Polysciences Inc.) and 690 μL of CHO-S-SFMII medium and allowed to stand for 10 minutes at room temperature. Incubated in a CO ₂ incubator (5% CO ₂ at 37 ° C.) for 4-5 hours. Then, 6.9 mL of CHO-S-SFM-II (Invitrogen) medium was added and cultured in a CO ₂ incubator for 3 days. After the culture supernatant was recovered, centrifuged (approximately 2000 g, 5 min, room temperature) to remove the cells, and sterilized through a further 0.22μm filter MILLEX ^(R) -GV (Millipore). The sample was stored at 4 ° C. until use.

Purification of IgG antibody 50 μL of rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) suspended in TBS was added to the obtained culture supernatant and mixed by inverting at 4 ° C. for 4 hours or more. The solution was transferred to a 0.22 μm filter cup Ultrafree ^(R) -MC (Millipore), washed 3 times with 500 μL of TBS, then suspended in 100 μL of 50 mM aqueous sodium acetate, pH 3.3 in rProtein A Sepharose ^™ resin. After standing for a minute, the antibody was eluted. Immediately, 6.7 μL of 1.5 M Tris-HCl, pH 7.8 was added for neutralization. Elution was performed twice to obtain 200 μL of purified antibody. Absorbance at 280 nm was measured by applying 2 μL of the antibody-containing solution to the ND-1000 Spectrophotometer (NanoDrop) or 50 μL to the spectrophotometer DU-600 (BECKMAN). The antibody concentration was calculated from the obtained value using the following formula.
Antibody concentration (mg / mL) = Absorbance x Dilution factor / 14.6 x 10

Evaluation of human IL-6 receptor neutralizing activity of IgG clones
IL-6 receptor neutralizing activity was evaluated using BaF3 / gp130 showing IL-6 / IL-6 receptor-dependent proliferation. After washing BaF3 / gp130 three times with RPMI1640 medium containing 10% FBS, 60 ng / mL human interleukin-6 (TORAY), 60 ng / mL recombinant soluble human IL to 5x10 ⁴ cells / mL Suspended in RPMI1640 medium containing -6 receptor (SR344) and 10% FBS, 50 μL was dispensed into each well of a 96 well-plate (CORNING). Next, the purified antibody was diluted with RPMI1640 containing 10% FBS, and 50 μL was mixed with each well. Add WST-8 reagent (Cell Counting Kit-8, Dojindo Laboratories, Inc.) diluted 2 times with PBS at 37 μC, 5% CO ₂ for 3 days at 20 μL / well. Absorbance at 450 nm (reference wavelength 620 nm) was measured using SUNRISE CLASSIC (TECAN). After culturing for 2 hours, the absorbance at 450 nm (reference wavelength 620 nm) was measured again, and IL-6 receptor neutralizing activity was evaluated using the change in absorbance for 2 hours as an index.
As a result, a plurality of antibodies having higher activity compared to the humanized PM1 antibody (wild type: WT) were obtained. FIG. 4 shows antibody mutation sites showing higher activity than WT. For example, as shown in FIG. 1, it was found that RD_6 exhibits neutralizing activity that is about 50 times higher than WT as a 100% inhibitory concentration.

Biacore T100 (BIACORE) was used to analyze the kinetics of antigen-antibody reaction for IgG clones whose Biacore affinity analysis activity was higher than that of the wild type. Immobilize rec-Protein A (ZYMED) (hereinafter Protein A) on the sensor chip from 1800RU to 2600RU, bind various antibodies to it, flow the antigen there as an analyte, and measure the interaction between antibody and antigen did. Recombinant human IL-6R sR (R & D systems) (hereinafter rhIL-6sR) adjusted to various concentrations was used as the antigen. All measurements were performed at 25 ° C. The association rate constant k _a (1 / Ms) and dissociation rate constant k _d (1 / s), which are kinetic parameters, are calculated from the sensorgram obtained by measurement, and K _D (M ) Was calculated. Biacore T100 Evaluation Software (BIACORE) was used for calculation of each parameter.
As a result, a plurality of antibodies having higher affinity than the humanized PM1 antibody (wild type: WT) were obtained. For example, sensorgrams of wild type (WT) and RD_6 are shown in FIGS. From the results of kinetic parameter analysis, it was found that RD_6 showed about 50 times higher affinity than WT (Table 1). Other antibodies with an affinity several tens of times higher were obtained. FIG. 4 shows mutation sites showing higher affinity than WT.

[Example 2] For mutation sites with improved activity and antigen affinity for antigen binding ability by combination of CDR modifications , mutation sites were fused to produce antibodies with higher activity and higher affinity.
Preparation, expression, and purification of modified antibody Amino acid modification was carried out to produce a modified antibody at selected locations. Specifically, using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), the H (WT) variable region (H (WT), base sequence number: 107) prepared by the method described in the attached instructions, and L A mutation was introduced into the (WT) chain variable region (L (WT), base sequence number: 108). After digesting the H chain antibody gene fragment insertion plasmid, which was confirmed to be the target humanized antibody variable region gene sequence, with XhoI and NotI, the L chain antibody gene fragment insertion plasmid was digested with EcoRI, It was subjected to% agarose gel electrophoresis. A DNA fragment of the desired size (about 400 bp) was purified using the QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction, and eluted with 30 μl of sterilized water. Thereafter, the H chain antibody gene fragment was inserted into an animal cell expression vector to prepare the desired H chain expression vector. Similarly, an L chain expression vector was prepared. For the ligation reaction, E. coli DH5α strain (Toyobo) was transformed using Rapid DNA Ligation Kit (Roche Diagnostics). The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the DNA sequencer ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached instructions. Expression and purification were performed by the method described in Example 1 using the prepared expression vector.

Evaluation of human IL-6 receptor neutralizing activity The neutralizing activity of the purified antibody was evaluated by the method shown in Example 1. However, human interleukin-6 (TORAY) concentration was 600 ng / mL, and neutralization activity was evaluated. A plurality of novel antibodies exhibiting higher activity than WT were obtained, and their CDR sequences are shown in FIG. Among them, FIG. 6 shows the neutralizing activity of an antibody (referred to as RDC_23) using RDC_5H for the H chain and RDC_11L for the L chain as an antibody exhibiting high activity. It became clear that RDC_23 has about 100 times higher activity as a 100% inhibitory concentration compared with WT. Not only RDC_23, which is an antibody using RDC_5H for H chain and RDC_11L for L chain, but also RDC_2H, RDC_3H, RDC_4H, RDC_5H, RDC_6H, RDC_7H, RDC_8H, RDC_27H, RDC_28H, RDC_29H, RDC_30H, RDC_30H, RDC_30H, RDC_30H Antibodies using L (WT) (respectively RDC_2, RDC_3, RDC_4, RDC_5, RDC_6, RDC_7, RDC_8, RDC_27, RDC_28, RDC_29, RDC_30, RDC_32), and H (WT), L chain in the H chain In addition, the antibody using RDC_11L (referred to as RDC_11) has been confirmed to have improved neutralizing activity, and by combining mutation sites found in affinity maturation, antibodies with higher neutralizing activity can be obtained. It became clear. Moreover, since the antibody combining these mutation sites has improved neutralizing activity, it was considered that affinity was also improved.

このことから、これらの抗体のアフィニティーは変異箇所の組み合わせ前の抗体よりも強いと考えられた。中和活性と同様、アフィニティーマチュレーションで見出された変異箇所を組み合わせることで、より高いアフィニティーを有する抗体が取得可能であることが明らかとなった。WTと比較して高い活性あるいはアフィニティーを示す変異体のアミノ酸配列は以下のとおりである（WTからの変異箇所に下線）。
（HCDR2）
配列番号：４５ YISYSGITNYNPSLKS
（HCDR3）
配列番号：５７ LLARATAMDY
配列番号：５８ VLARATAMDY
配列番号：５９ ILARATAMDY
配列番号：６０ TLARATAMDY
配列番号：６１ VLARITAMDY
配列番号：６２ ILARITAMDY
配列番号：６３ TLARITAMDY
配列番号：６４ LLARITAMDY
（LCDR3）
配列番号：７９ GQGNRLPYT
すなわち、HCDR2の９番目のアミノ酸がAsnであり、HCDR3の1番目のアミノ酸がLeu、Val、Ile、Thrのいずれかから選択され、HCDR3の5番目のアミノ酸がAla、Ileのいずれかから選択され、LCDR3の1番目のアミノ酸がGlyであり、LCDR3の5番目のアミノ酸がArgである抗体を作製することで、WTよりも中和活性、およびアフィニティーが著しく向上した抗IL-6レセプター抗体を作製することが可能である。 Affinity analysis by Biacore using ProteinA Therefore, among antibodies with improved neutralization activity, RDC_2, RDC_3, RDC_4, RDC_5, RDC_6, RDC_7, RDC_8, RDC_11, and RDC_23 were tested for antigen-antibody reaction A kinetic analysis was performed. Immobilize rec-Protein A (ZYMED) from 4400 RU to 5000 RU on the sensor chip by the amine coupling method, bind various antibodies to it, and flow the antigen as an analyte to detect the interaction between the antibody and antigen. It was measured. As the antigen, rhIL-6sR adjusted to various concentrations was used. All measurements were performed at 25 ° C. From the sensorgram obtained from the measurement, the association rate constant k _a (1 / Ms) and dissociation rate constant k _d (1 / s), which are kinetic parameters, are calculated, and K _D (M ) Was calculated. Biacore T100 Evaluation Software (BIACORE) was used for calculation of each parameter. As a result, a combination of mutation sites RDC_2, RDC_3, RDC_4, RDC_5, RDC_6, RDC_7, RDC_8, RDC_11, ( tables mutation locations were measured simultaneously had a smaller K _D values than RD_28 is one location in RDC_23 2). Among them, a sensorgram is shown in FIG. 7 for RDC_23 which showed high affinity.

From this, it was considered that the affinity of these antibodies was stronger than that of the antibody before the combination of mutation sites. Similar to the neutralizing activity, it was revealed that antibodies having higher affinity can be obtained by combining mutation sites found by affinity maturation. The amino acid sequences of the mutants showing higher activity or affinity than WT are as follows (underlined mutation sites from WT).
(HCDR2)
SEQ ID NO: 45 YISYSGIT N YNPSLKS
(HCDR3)
SEQ ID NO: 57 L LAR A TAMDY
SEQ ID NO: 58 V LAR A TAMDY
SEQ ID NO: 59 I LAR A TAMDY
SEQ ID NO: 60 T LAR A TAMDY
SEQ ID NO: 61 V LAR I TAMDY
SEQ ID NO: 62 I LAR I TAMDY
SEQ ID NO: 63 T LAR I TAMDY
SEQ ID NO: 64 L LAR I TAMDY
(LCDR3)
SEQ ID NO: 79 G QGN R LPYT
That is, the ninth amino acid of HCDR2 is Asn, the first amino acid of HCDR3 is selected from any of Leu, Val, Ile, or Thr, and the fifth amino acid of HCDR3 is selected from any of Ala or Ile. By producing an antibody in which the first amino acid of LCDR3 is Gly and the fifth amino acid of LCDR3 is Arg, an anti-IL-6 receptor antibody with significantly improved neutralizing activity and affinity than WT is produced. Is possible.

Affinity analysis with Biacore using Protein A / G
Using Biacore T100 (BIACORE), kinetic analysis of antigen-antibody reaction of WT and RDC_23 was performed. Immobilize Purified Recomb Protein A / G (Pierce) (hereinafter referred to as Protein A / G) on the sensor chip, bind various antibodies to it, flow the antigen there as an analyte, and investigate the interaction between the antibody and antigen. It was measured. As the antigen, rhIL-6sR (R & D systems) adjusted to various concentrations and recombinant soluble IL-6 receptor (SR344 prepared in Example 1) were used. The rhIL-6sR produced from insect cells infected with baculovirus has a high mannose type sugar chain structure, whereas SR344 produced from CHO cells has a complex type sugar chain with sialic acid at the end. It is thought that it is united. Since the actual sugar chain structure of soluble IL-6 receptor in the human body is a complex sugar chain and sialic acid is bound to the terminal, SR344 is more soluble in human body. In this experiment, we compared rhIL-6sR and SR344.
The association rate constant k _a (1 / Ms) and dissociation rate constant k _d (1 / s), which are kinetic parameters, are calculated from the sensorgram obtained by measurement, and K _D (M ) Was calculated. Biacore T100 Evaluation Software (BIACORE) was used for calculation of each parameter.
The sensor chip was prepared by immobilizing Protein A / G on CM5 (BIACORE) at approximately 3000 RU by the amine coupling method. Using the prepared sensor chip, kinetic analysis of the interaction between protein A / G-bound antibody (WT and RDC_23) and two soluble IL-6 receptors, rhIL-6sR and SR344 was performed. It was. The running buffer was HBS-EP +, and the flow rate was 20 μL / min. Each antibody was prepared to bind approximately 100 RU to Protein A / G. The rhIL-6sR used as the analyte was adjusted to 0, 0.156, 0.313, and 0.625 μg / mL using HBS-EP +, and SR344 was adjusted to 0, 0.0654, 0.131, and 0.261 μg / mL. First, the target antibodies WT and RDC_23 were bound to Protein A / G, the analyte solution was allowed to interact there for 3 minutes, then switched to HBS-EP + (BIACORE) and the dissociation phase was measured for 10 minutes. After measurement of the dissociated phase, the sensor chip was regenerated by washing with 10 uL of 10 mM glycine-HCl (pH 1.5). This binding / dissociation / regeneration was defined as one cycle of analysis. All experiments were performed at 37 ° C.
WT and RDC_23 were measured according to this cycle, and the sensorgrams obtained for the two soluble IL-6 receptors, rhIL-6sR and SR344, are shown in FIG. 8, FIG. 9, FIG. 10, and FIG. It was. The obtained sensorgram was subjected to kinetic analysis using Biacore T100 Evaluation Software, which is data analysis software dedicated to Biacore (Table 3). As a result, in comparison between rhIL-6sR and SR344, it was revealed that the affinity obtained using SR344 for both WT and RDC_23 was weakened by 2-3 times. RDC_23 has an affinity that is about 40 to 60 times higher than that of WT for both rhIL-6sR and SR344, and it is soluble in the human body by the combination of each CDR modification obtained by affinity maturation. It was also revealed that SR344, which is considered to be close to the structure of the type IL-6 receptor, has a very strong affinity compared to WT. In the following examples, all measurements are performed at 37 ° C., and kinetic analysis of the antigen-antibody reaction using SR344 and protein A / G is performed.

[Example 3] Creation of H53 / L28 with improved plasma retention and reduced immunogenicity risk by modifying CDR and framework
Cancer Res. 1993 Feb 15; 53 (4): 851-6 Humanized mouse PM1 antibody (hereinafter abbreviated as Wild type, WT, H chain WT is H (WT), L chain WT is L (WT) The following modifications were made with the aim of improving plasma retention, reducing immunogenicity risk, and improving stability. In order to improve the plasma retention, a modification that lowers the isoelectric point was introduced into the WT H chain variable region and L chain variable region sequences.

Preparation of three-dimensional structure model of humanized PM1 antibody First, MOE software (Chemical Computing Group Inc.) was used to confirm the amino acid residues exposed on the variable region surface of humanized PM1 antibody (H (WT) / L (WT)). ) Was used to create a mouse PM1 antibody Fv region model that was humanized by homology modeling.

Selection of the modified site for lowering the isoelectric point of the humanized PM1 antibody By detailed analysis of the prepared model, the FR sequence as an introduction site for modification that lowers the isoelectric point, among the amino acids exposed on the surface, H16, H43, H81, H105, L18, L45, L79, L107 (Kabat numbering, Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH) have CDR sequences of H31, H64, H65, L24, L27, L53. , L55 was considered to be a candidate for reducing the isoelectric point without reducing activity or stability.

Removal of mouse sequence remaining in humanized PM1 antibody Humanized PM1 antibody is an antibody sequence obtained by humanizing mouse PM1 antibody (Cancer Res. 1993 Feb 15; 53 (4): 851-6). The H chain of the humanized PM1 antibody is CDR-grafted in the framework of NEW, which is a human antibody variable region, but the H27, H28, H29, H30, and H71 of the H chain use mouse sequences as they are to maintain activity. is doing. Considering the risk of immunogenicity, the smaller the mouse sequence, the better. Therefore, we searched for a sequence to make H27, H28, H29 and H30 human sequences.

Selection of modified sites to improve the stability of humanized PM1 antibody Substitution of H65 from serine to glycine (stabilization of turn structure) in the variable region of humanized PM1 antibody (H (WT) / L (WT)) Stabilization by modification to the consensus sequence of HCDR2), substitution of H69 from methionine to isoleucine (stabilizes the hydrophobic core structure), and substitution of H70 from leucine to serine (to hydrophilize surface exposed residues) ), Substitution of H58 from threonine to asparagine (stabilized by modification to the consensus sequence of HCDR2), substitution of L93 from threonine to serine (stabilized by hydrophilizing surface exposed residues) ), It was considered possible to improve the stability by performing isoleucine (stabilization of β-sheet) from serine of H107, and these modifications were considered candidates for improving the stability.

Removal of T-cell epitope predicted by in silico of humanized PM1 antibody Introduction The variable region of humanized PM1 antibody (H (WT) / L (WT)) was changed to TEPITOPE (Methods. 2004 Dec; 34 (4): 468 -75). As a result, it was shown that L-chain CDR2 has many T-cell epitopes that bind to HLA. Therefore, TEPITOPE analysis examined modifications that did not reduce stability, binding activity, and neutralization activity while reducing the immunogenicity risk of L chain CDR2. As a result, it was clarified that by replacing threonine of L51 of L chain CDR2 with glycine, the T-cell epitope bound to HLA can be removed without reducing stability, binding activity, and neutralizing activity.

また、上述のH53のFR3は非ヒト配列が存在しているため、さらに免疫原性リスクを低減することが望ましい。免疫原性リスクを低減する改変として、H89のAlaをValに置換した配列（配列番号：１２７）にすることが可能と考えられる。さらにH53のFR3のH71のArgは結合活性に重要であるため（Cancer Res. 1993 Feb 15;53(4):851-6）、H71がArgで保存されているヒトVH1サブクラスのFR3配列（配列番号：１２８）、あるいは、ヒトVH3サブクラスのFR3配列（配列番号：１２９）を用いることで、H鎖、L鎖ともにフレームワークとしては完全にヒト配列である抗ヒトIL-6レセプター抗体を作製可能であると考えられる。 Selection of each framework sequence Human from the publicly available Kabat Database (ftp://ftp.ebi.ac.uk/pub/databases/kabat/) and IMGT Database (http://imgt.cines.fr/) By obtaining antibody amino acid sequence data and using the constructed database, homology search can be performed separately for each frame. In selecting a human framework, human framework sequences having the above-mentioned modifications were examined from a database from the viewpoints of lowering the isoelectric point, removing the remaining mouse sequences, and improving stability. As a result, it has been clarified that the following items can be satisfied without reducing the binding activity and the neutralizing activity by making each framework of the modified antibody H53 / L28 into the following sequences as shown below. Source is derived from the human sequence, and the amino acid residue in the underlined part of the sequence is an amino acid introduced with a modification compared with WT.

In addition, since the FR3 of H53 described above has a non-human sequence, it is desirable to further reduce the immunogenicity risk. As a modification that reduces the risk of immunogenicity, it is considered possible to use a sequence (SEQ ID NO: 127) in which A89 of H89 is replaced with Val. Furthermore, since the Ar of H71 FR3 of H53 is important for binding activity (Cancer Res. 1993 Feb 15; 53 (4): 851-6), the FR3 sequence of the human VH1 subclass in which H71 is conserved in Arg (sequence) No. 128), or by using the human VH3 subclass FR3 sequence (SEQ ID NO: 129), it is possible to produce anti-human IL-6 receptor antibodies that are completely human sequences as the framework of both H and L chains. It is thought that.

Selection of each CDR sequence
The CDR sequences of H53 / L28 are as follows from the viewpoint of lowering the isoelectric point, improving stability, and removing T-cell epitopes without first reducing the binding activity and neutralization activity. Selected.

Preparation, expression and purification of modified antibody expression vector Expression vector preparation, expression and purification of the modified antibody were carried out by the method described in Example 1. Modifications were sequentially introduced so that the framework sequences and CDR sequences selected for the humanized mouse PM1 antibody H (WT) mutagenesis vector, L (WT) mutagenesis vector were selected. The animal cell expression vector encoding the finally obtained defined framework sequence, H53 / L28 (antibody amino acid sequence H53 SEQ ID NO: 104, L28 SEQ ID NO: 105) having a CDR sequence, L28 was expressed and purified and used for the following evaluation.

Isoelectric point evaluation of modified antibody H53 / L28 by isoelectric focusing To evaluate the change in isoelectric point of full-length antibody due to amino acid modification of variable region, by isoelectric focusing of WT and modified antibody H53 / L28 Analysis was performed. Isoelectric focusing was performed as follows. Phast-Gel Dry IEF (Amersham Biosciences) gel was swollen for about 30 min with the following swelling solution using Phastsystem Cassette (Amersham Biosciences).
Milli-Q water 1.5 mL
Pharmalyte 5-8 for IEF (Amersham Biosciences) 50μL
Pharmalyte 8-10.5 for IEF (Amersham Biosciences) 50μL
Using the swollen gel, electrophoresis was performed by PhastSystem (manufactured by Amersham Biosciences) with the following program. The sample was added to the gel in Step 2. As a pI marker, Calibration Kit for pI (Amersham Biosciences) was used.
Step 1: 2000 V 2.5 mA 3.5 W 15 ° C 75 Vh
Step 2: 200 V 2.5 mA 3.5 W 15 ° C 15 Vh
Step 3: 2000 V 2.5 mA 3.5 W 15 ° C 410 Vh
The gel after electrophoresis was fixed with 20% TCA, and then silver staining was performed using Silver staining Kit, protein (Amersham Biosciences) according to the protocol attached to the kit. After staining, the isoelectric point of the sample (full-length antibody) was calculated from the known isoelectric point of the pI marker. As a result, the isoelectric point of WT is about 9.3, the isoelectric point of the modified antibody H53 / L28 is about 6.5 to 6.7, and H53 / L28 having an isoelectric point reduced by about 2.7 by amino acid substitution is obtained from WT. It was. Further, when the theoretical isoelectric point of the variable region (VH, VL sequence) of H53 / L28 was calculated by GENETYX (GENETYX CORPORATION), the theoretical isoelectric point was 4.52. Since the theoretical isoelectric point of WT is 9.20, H53 / L28 in which the theoretical isoelectric point of the variable region was reduced by about 4.7 by amino acid substitution from WT was obtained.

Evaluation of human IL-6 receptor neutralizing activity of H53 / L28
WT and H53 / L28 were carried out by the method shown in Example 1. The results are shown in FIG. As a result, it was revealed that the modified antibody H53 / L28 has several times higher neutralization activity of BaF / gp130 than WT. That is, H53 / L28 was able to improve neutralizing activity while lowering the isoelectric point compared to WT.

Affinity analysis of H53 / L28 for human IL-6 receptor by Biacore
The affinity of WT and H53 / L28 to human IL-6 receptor was analyzed by kinetic analysis using Biacore T100 (BIACORE). Immobilize Purified Recomb Protein A / G (Pierce) (hereinafter referred to as Protein A / G) on the sensor chip, bind various antibodies to it, flow the antigen there as an analyte, and investigate the interaction between the antibody and antigen. It was measured. As the antigen, recombinant soluble IL-6 receptor (SR344) adjusted to various concentrations was used. The measurement conditions were the same as in Example 2.
The obtained sensorgrams of WT and H53 / L28 are shown in FIG. Table 6 shows the results of kinetic analysis using Biacore T100 Evaluation Software, a data analysis software dedicated to Biacore. Consequently, H53 / L28 has declined K _D of approximately 6-fold compared to WT, it was found to affinity is improved by about 6 fold. That is, H53 / L28 was able to improve the affinity about 6 times while lowering the isoelectric point compared with WT. As a result of detailed examination, it was considered that the amino acid mutation contributing to the improvement of affinity was a mutation in which threonine of L51 was replaced with glycine. That is, it was thought that affinity could be improved by replacing threonine of L51 with glycine.

T-cell epitope prediction by TEPITOPE of H53 / L28
TEPITOPE analysis of H53 / L28 (Methods. 2004 Dec; 34 (4): 468-75) was performed. As a result, it was found that the number of peptides that could bind to HLA was significantly reduced compared to WT, and it was considered that the risk of immunogenicity in humans was reduced.

H53/L28の静脈内投与後の血漿中半減期(T1/2)はWTの約1.3倍に延長し、クリアランスが約1.7倍低下した。H53/L28の皮下投与後のT1/2はWTの約2倍に延長し、クリアランスが約2.1倍低下した。このようにWTの等電点を低下させることによって、H53/L28の血漿中滞留性を大幅に向上させることが出来た。
H53/L28は、ヒト化PM-1抗体（WT）と比較して、結合活性、中和活性を向上させ、免疫原性リスクを低減させつつ、血漿中滞留性を大幅に向上させたヒト化抗IL-6レセプター抗体であることから、医薬品として開発する上でH53/L28で適用した改変は極めて有用であると考えられた。 [Example 4] Evaluation of plasma retention of H53 / L28
In order to evaluate the plasma retention of the modified antibody H53 / L28 with a reduced isoelectric point in order to evaluate the plasma retention of the modified antibody H53 / L28 in normal mice A comparison was made.
WT and H53 / L28 were administered to mice (C57BL / 6J, Charles River, Japan) as a single dose of 1 mg / kg intravenously and subcutaneously before and after administration for 15 minutes, 2 hours, 8 hours, 1 day, 2 days. Blood was collected for 5, 7, 14, 21, 21, and 28 days. However, blood was collected from only the intravenous administration group for 15 minutes after administration. The collected blood was immediately centrifuged at 4 ° C. and 15,000 rpm for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set to −20 ° C. or lower until measurement was performed.
Mouse plasma concentration was measured by ELISA. First, Recombinant Human IL-6 sR (R & D Systems) was biotinized using EZ-Link ™ Sulfo-NFS-Biotinylation Kit (PIERCE). Distribute this biotinylated human-sIL-6R to Reacti-Bind Streptavidin High Binding Capacity (HBC) Coated Plates (manufactured by PIERCE) and let it stand at room temperature for 1 hour or more to create a human-sIL-6R solid-phase plate did. Prepare calibration curve samples and mouse plasma measurement samples with plasma concentrations of 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05 μg / mL and dispense them on human-sIL-6R solid-phase plates for 1 hour at room temperature Left to stand. Anti-human IgG-AP (manufactured by SIGMA) was then reacted, color reaction was performed using BluePhos Microwell Phosphatase Substrates System (Kirkegaard & Perry Laboratories) as a substrate, and absorbance at 650 nm was measured with a microplate reader. . The mouse plasma concentration was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices). FIG. 14 shows changes in plasma concentration after intravenous administration of WT and H53 / L28, and FIG. 15 shows changes in plasma concentration after subcutaneous administration.
Non-model-dependent analysis of the obtained plasma concentration transition data with the pharmacokinetic analysis software WinNonlin (Pharsight) and pharmacokinetic parameters (AUC, clearance (CL), half-life (T1 / 2)) Calculated. T1 / 2 was calculated from the plasma concentration of the final three points or the final phase automatically set by WinNonlin. BA was calculated from the ratio of AUC after subcutaneous administration to AUC after intravenous administration. The obtained pharmacokinetic parameters are shown in Table 7.

The plasma half-life (T1 / 2) after intravenous administration of H53 / L28 was about 1.3 times that of WT, and the clearance was reduced about 1.7 times. T1 / 2 after subcutaneous administration of H53 / L28 was extended to about twice that of WT, and the clearance was reduced about 2.1 times. Thus, by reducing the isoelectric point of WT, the plasma retention of H53 / L28 could be significantly improved.
H53 / L28 has improved binding and neutralization activities compared to humanized PM-1 antibody (WT), reducing the risk of immunogenicity, and significantly increasing plasma retention Since it is an anti-IL-6 receptor antibody, the modification applied with H53 / L28 was considered to be extremely useful in developing it as a pharmaceutical product.

[Example 5] Preparation of PF1 antibody
Production of humanized PM1 antibody expression vector and mutagenesis vector H53 / L28 prepared in Example 4 improves the affinity of RDC_23 found in Example 2 at 2 H chains and 2 L chains for a total of 4 sites A CDR mutation was introduced. The H chain with the RDC_23 mutation introduced into H53 / L28 is designated as PF1_H, and the L chain with the RDC_23 mutation introduced into H53 / L28 is designated as PF1_L, and a modified antibody is produced, expressed and purified by the method described in Example 1. did. The amino acid sequence of PF1_H is shown in SEQ ID NO: 22, and the amino acid sequence of PF1_L is shown in SEQ ID NO: 23.

Evaluation of human IL-6 receptor neutralization activity The neutralization activity of the purified PF1 antibody was evaluated by the method shown in Example 1. However, human interleukin-6 (TORAY) concentration was 600 ng / mL, and neutralization activity was evaluated. The neutralizing activity of WT and PF1 is shown in FIG. It was revealed that PF1 has an activity of about 100 to 1000 times as 100% inhibitory concentration compared with WT.

Affinity analysis of PF1 antibody for human IL-6 receptor by Biacore This measurement was performed under the same conditions as in Example 2. The running buffer was HBS-EP +, and the flow rate was 20 μL / min. Each antibody was prepared to bind approximately 100 RU to Protein A / G. SR344 used as an analyte was adjusted to 0, 0.065, 0.131, 0.261 μg / mL using HBS-EP +. First, the antibody solution was bound to Protein A / G, the analyte solution was allowed to interact there for 3 minutes, then switched to HBS-EP + and the dissociation phase was measured for 10 or 15 minutes. After measurement of the dissociated phase, the sensor chip was regenerated by washing with 10 μL of 10 mM glycine-HCl (pH 1.5). This binding / dissociation / regeneration was defined as one cycle of analysis. Various antibodies were measured according to this cycle.
The obtained sensorgram of PF1 is shown in FIG. Table 8 shows the results of kinetic analysis of the obtained sensorgrams using Biacore T100 Evaluation Software, which is Biacore's exclusive data analysis software, together with the results of WT and H53 / L28. As a result, it was revealed that the affinity of PF1 was improved about 150 times compared with WT. By combining RDC_23 with high affinity obtained by the combination of affinity maturation and H53 / L28 with improved plasma retention and affinity, the affinity of PF1 is higher than these due to the additive effect. was gotten.

Thermal stability evaluation of PF1 antibody by differential scanning calorimetry (DSC)
In order to evaluate the thermal stability of PF1 antibody, thermal denaturation intermediate temperature (Tm value) was evaluated by differential scanning calorimetry (DSC). Dialyze (EasySEP, TOMY) the purified antibody of WT and PF1 against a solution of 20 mM sodium acetate, 150 mM NaCl, pH 6.0, and at a protein concentration of about 0.1 mg / mL, 1 ° C / 100 ° C from 40 ° C to 100 ° C DSC measurement was performed at a heating rate of min. As a result, it was found that the Tm value of the Fab portion of WT was about 94 ° C., and the Tm value of the Fab portion of PF1 was 91 ° C. Since the Tm value of the Fab part of a general IgG1-type antibody molecule is in the range of about 60 ° C. to 85 ° C. (Biochem Biophys Res Commun. 2007 Apr 13; 355 (3): 751-7., Mol Immunol. 2007 Apr; 44 (11): 3049-60), and the thermal stability of the obtained PF1 antibody was shown to be extremely high compared to general IgG1 molecules.

High concentration stability evaluation of PF1 antibody
The stability of PF1 antibody in high concentration preparations was evaluated. The purified antibody of WT and PF1 was dialyzed (EasySEP, TOMY) against a solution of 20 mM histidine chloride, 150 mM NaCl, pH 6.5, then concentrated with an ultrafiltration membrane, and a high concentration stability test was performed. The conditions are as follows.
Antibodies: WT and PF1
Buffer: 20 mM histidine chloride, 150 mM NaCl, pH 6.0
Concentration: 145 mg / mL
Storage temperature and duration: 25 ° C-2 weeks, 25 ° C-4 weeks, 25 ° C-7 weeks Assess method: System Waters Alliance
Column G3000SWxl (TOSOH)
Mobile phase 50 mM sodium phosphate, 300 mM KCl, pH 7.0
Flow velocity / wavelength 0.5ml / min, 220nm
Dilute sample to 1/100 for analysis
The aggregate content of the preparation after storage at initial (immediately after preparation of the preparation) and after storage under each condition was evaluated by the gel filtration chromatography described above, and the amount of change (increase) in the aggregate content from the initial is shown in FIG. As a result, both WT and PF1 showed very high stability, and it was found that the increased amount of aggregates at 25 ° C-7 weeks was about 0.7% for WT and about 0.3% for PF1, respectively. The increase in aggregates per month was about 0.4% and about 0.17%, respectively, indicating that PF1 exhibits extremely high stability, especially at high concentrations. In WO 2003/039485, stability data at 25 ° C of Daclizumab 100mg / mL formulation already marketed as a high-concentration IgG preparation is shown. The increase in aggregates is about 0.3%, and PF1 is extremely superior in stability at high concentrations compared to Daclizumab. In developing a high-concentration solution formulation, the increase in aggregates is large. Although it is a problem, it was shown that PF1 antibody showed very little increase in aggregates at high concentrations.
PF1 improves antigen binding capacity, improves plasma retention by lowering the isoelectric point, and reduces immunogenicity risk by removing residual mouse sequences and removing T-cell epitopes, stability over WT Although it is a molecule that has been modified for the purpose of improving its function, it has become clear that the stability in high-concentration preparations of 100 mg / mL or higher is actually very high compared to WT. It is possible to provide a stable and convenient high-concentration subcutaneous preparation.

[Example 6] PK / PD test of PF1 antibody in human IL-6 receptor transgenic mice
Pharmacokinetic study using human IL-6 receptor transgenic mice
About WT and PF1 prepared in Example 5, pharmacokinetics in human IL-6 receptor transgenic mice (hIL-6R tg mice, Proc Natl Acad Sci US A. 1995 May 23; 92 (11): 4862-6) The in vivo neutralizing ability of human soluble IL-6 receptor was evaluated. WT and PF1 were administered to hIL-6R tg mice at a single dose of 10 mg / kg intravenously for 15 minutes, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 4 days, 7 days after administration. Blood was collected. The collected blood was immediately centrifuged at 4 ° C. and 15,000 rpm for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set to −20 ° C. or lower until measurement was performed.
Mouse plasma concentration was measured by ELISA. Calibration curve samples having plasma concentrations of 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 μg / mL were prepared. An immunoplate (Nunc-Immuno Plate, MaxiSorp (Nalge nunc International) manufactured by immobilizing calibration curve samples and mouse plasma measurement samples with Anti-human IgG (γ-chain specific) F (ab ') 2 (Sigma) )) And allowed to stand at room temperature for 1 hour, and then sequentially reacted with Goat Anti-Human IgG-BIOT (manufactured by Southern Biotechnology Associates) and Streptavidin-alkaline phosphatase conjugate (manufactured by Roche Diagnostics), and BluePhos Microwell Phosphatase Substrates A color reaction was performed using System (Kirkegaard & Perry Laboratories) as a substrate, and the absorbance at 650 nm was measured with a microplate reader. The mouse plasma concentration was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices). Changes in plasma concentrations of WT and PF1 are shown in FIG. PF1 increased plasma retention in human IL-6 receptor transgenic mice compared to WT, as the plasma concentration 4 days after administration of PF1 was about 5 times higher than WT. It became clear that.
Human IL-6 receptor transgenic mice have been shown to produce human soluble IL-6 receptor in plasma. Therefore, it is possible to evaluate the neutralizing effect of human soluble IL-6 receptor present in plasma by administering an anti-human IL-6 receptor antibody to human IL-6 receptor transgenic mice.
In order to evaluate the degree of neutralization of human soluble IL-6 receptor by administration of WT or PF1, measurement of the concentration of unbound human soluble IL-6 receptor in mouse plasma was measured. did. 6 μL of mouse plasma is diluted 2-fold with dilution buffer containing BSA and added to the appropriate amount of rProtein A Sepharose Fast Flow (GE Healthcare) resin dried in a 0.22 μm filter cup (Millipore). All the IgG type antibodies present (mouse IgG, anti-human IL-6 receptor antibody and anti-human IL-6 receptor antibody-human soluble IL-6 receptor complex) were adsorbed to proteinA. Then, it spin-down with the high-speed centrifuge and collect | recovered pass solutions. Since the path solution does not contain anti-human IL-6 receptor antibody-human soluble IL-6 receptor complex bound to protein A, the concentration of human soluble IL-6 receptor in the path solution can be determined by measuring It is possible to measure the concentration of unbound soluble IL-6 receptor. As the soluble IL-6 receptor concentration, Quantikine Human IL-6 sR (R & D Systems) was used. Measurement of unbound soluble IL-6 receptor concentration after 4 hours, 8 hours, 24 hours, 48 hours, 96 hours, and 168 hours of WT and PF1-treated mice according to the attached instructions did.
The results are shown in FIG. The concentration of soluble IL-6 receptor that is non-binding for both WT and PF1 is 10 ng / mL or less after 4 and 8 hours after a single intravenous dose of WT and PF1 at 10 mg / kg. It was confirmed that the soluble IL-6 receptor was neutralized. However, WT non-binding soluble IL-6 receptor concentration after 24 hours was about 500 ng / mL, whereas PF1 non-binding soluble IL-6 receptor concentration was 10 ng / mL. Since it was less than mL, it was found that PF1 neutralized human soluble IL-6 receptor more continuously than WT.
PF1 is a combination of RDC_23 found in affinity maturation and H53 / L28 with improved plasma retention, etc., and exhibits long plasma retention and high neutralizing activity in vivo. It was considered possible. In fact, in human IL-6 receptor transgenic mice producing human soluble IL-6 receptor, PF1 has been shown to have a neutralizing effect and plasma concentration that lasts longer than WT.
PF1 is superior to WT (humanized PM-1 antibody) in terms of stability and immunogenicity in high-concentration preparations, and also in the IL-6 receptor neutralizing effect and plasma in human IL-6 receptor transgenic mice Because of its excellent retention in the medium, the modification applied with PF1 was considered to be extremely useful for development as a pharmaceutical product.

[Example 7] Improvement of stability of IgG2 and IgG4 under acidic conditions
Production and expression of IgG2 and IgG4 humanized IL-6 receptor antibody expression vector
A molecule in which the constant region of the humanized PM1 antibody (Cancer Res. 1993 Feb 15; 53 (4): 851-6) is an IgG1 isotype, but the constant region is replaced with IgG2 in order to reduce the binding activity to the Fcγ receptor (WT-IgG2, SEQ ID NO: 109) and a molecule (WT-IgG4, SEQ ID NO: 110) substituted with IgG4 (Mol Immunol. 1993 Jan; 30 (1): 105-8.) Were prepared. Animal cell expression vectors were used for IgG expression. An expression vector was constructed in which the constant region was substituted with IgG2 or IgG4 by NheI / NotI digestion and ligation of the constant region of the humanized PM1 antibody (IgG1) used in Example 1. The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the DNA sequencer ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached instructions. WT was used as the L chain, and WT-IgG1, WT-IgG2, and WT-IgG4 were expressed by the method described in Example 1.
(1) Humanized PM1 antibody (WT-IgG1) H chain: SEQ ID NO: 15 (amino acid sequence)
(2) WT-IgG2 heavy chain: SEQ ID NO: 109 (amino acid sequence)
(3) WT-IgG4 heavy chain: SEQ ID NO: 110 (amino acid sequence)

Purification by protein A hydrochloric acid elution of WT-IgG1, WT-IgG2, and WT-IgG4 50 μL of rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) suspended in TBS was added to the obtained culture supernatant, and 4 ° C. Invert and mix for at least 4 hours. The solution was transferred to a 0.22 μm filter cup Ultrafree ^(R) -MC (Millipore), washed 3 times with 500 μL of TBS, suspended in 100 μL of 10 mM HCl, 150 mM NaCl, pH 2.0 in rProtein A Sepharose ^TM resin. After standing for 2 minutes, the antibody was eluted (hydrochloric acid elution method). Immediately, 6.7 μL of 1.5 M Tris-HCl, pH 7.8 was added for neutralization. Elution was performed twice to obtain 200 μL of purified antibody.

Gel filtration chromatography analysis of WT-IgG1, WT-IgG2, and WT-IgG4 purified by the hydrochloric acid elution method Gel filtration chromatography analysis was performed to evaluate the aggregate content of purified products obtained by the hydrochloric acid elution method.
Aggregate Evaluation Method: System Waters Alliance
Column G3000SWxl (TOSOH)
Mobile phase 50 mM sodium phosphate, 300 mM KCl, pH 7.0
Flow velocity / wavelength 0.5ml / min, 220nm
The results are shown in FIG. The aggregate content after purification of WT-IgG1 was about 2%, whereas the aggregate content after purification of WT-IgG2 and WT-IgG4 was about 25%. From this, IgG1 is stable to the acid at the time of hydrochloric acid elution, but IgG2 and IgG4 are unstable to the acid at the time of hydrochloric acid elution, and it is considered that denaturation / association has progressed. It was revealed that the stability under acidic conditions was lower than that of IgG1. In purification of IgG molecules, protein A is frequently used, but elution of IgG molecules from protein A is performed under acidic conditions. Virus inactivation necessary for developing IgG molecules as pharmaceuticals is generally performed under acidic conditions. From these facts, it is desirable that IgG molecules have higher stability under acidic conditions, but IgG2 and IgG4 molecules are found to be inferior to IgG1 in stability under acidic conditions. It became clear for the first time that there was a problem of denaturation and association. Although it was considered desirable to solve the problem of denaturation / association for development as a pharmaceutical, no method for solving this by amino acid substitution has been reported so far.

Production and evaluation of CH3 domain variants of WT-IgG2 and WT-IgG4
Since IgG2 and IgG4 molecules were shown to be less stable under acidic conditions than IgG1, variants that improve the stability of IgG2 and IgG4 molecules under acidic conditions were investigated. From the model of the constant region of IgG2 and IgG4 molecules, the instability of the CH3 domain in the CH3 domain can be considered as an instability factor under acidic conditions. As a result of various investigations, the 397th EU numbering in IgG2 In methionine and IgG4, the 409th arginine of EU numbering was thought to destabilize the CH3 / CH3 interface of IgG2 and IgG4, respectively. Therefore, an antibody in which the 397th methionine in the EU numbering of IgG2 is modified to valine (IgG2-M397V SEQ ID NO: 111 (amino acid sequence), and an antibody in which the 409th arginine in the EU numbering of IgG4 is modified to lysine (IgG4- R409K SEQ ID NO: 112 (amino acid sequence) was prepared.
Preparation, expression, and purification of the target antibody expression vector were performed using the hydrochloric acid elution method described above. Gel filtration chromatography analysis was performed to evaluate the aggregate content of the purified product obtained by the hydrochloric acid elution method from Protein A.
Aggregate Evaluation Method: System Waters Alliance
Column G3000SWxl (TOSOH)
Mobile phase 50 mM sodium phosphate, 300 mM KCl, pH 7.0
Flow velocity / wavelength 0.5ml / min, 220nm
The results are shown in FIG. The aggregate content after purification of WT-IgG1 was about 2%, whereas the aggregate content after purification of WT-IgG2 and WT-IgG4 was about 25%. On the other hand, the aggregate content of IgG2-M397V and IgG4-R409K, which are CH3 domain variants, was about 2%, the same level as IgG1. Improving the stability of IgG2 and IgG4 antibodies under acidic conditions by changing the 397th methionine in the EU numbering of IgG2 to valine, or by changing the 409th arginine of the EU numbering of IgG4 to lysine It became clear that it was possible to make it. Moreover, as a result of measuring the heat denaturation intermediate temperature of WT-IgG2, WT-IgG4, IgG2-M397V, and IgG4-R409K by the same method as in Example 5, IgG2- M397V and IgG4-R409K were each found to have a high Tm value in the CH3 domain into which the modification was introduced. From this, it was found that IgG2-M397V and IgG4-R409K are superior in thermal stability as compared with WT-IgG2 and WT-IgG4, respectively.
Since IgG2 and IgG4 are exposed to acidic conditions in the purification process using protein A and the virus inactivation process, denaturation / association in the same process was a problem, but as a constant region sequence of IgG2 and IgG4, It became clear that using IgG2-M397V and IgG4-R409K could solve the problem, and the modification proved to be extremely useful in developing IgG2 and IgG4 antibodies as pharmaceuticals. It was. In addition, IgG2-M397V and IgG4-R409K were found to be useful from the viewpoint of excellent thermal stability.

[Example 8] Improvement of heterogeneity derived from disulfide bond of IgG2
Purification of protein WT-IgG1, WT-IgG2, and WT-IgG4 by elution with protein A acetic acid 50 μL of rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) suspended in TBS was added to the culture supernatant obtained in Example 7. Then, it was mixed by inverting at 4 ° C for 4 hours or more. The solution was transferred to a 0.22 μm filter cup Ultrafree ^(R) -MC (Millipore), washed 3 times with 500 μL of TBS, then suspended in 100 μL of 50 mM aqueous sodium acetate, pH 3.3 in rProtein A Sepharose ^™ resin. After standing for a minute, the antibody was eluted. Immediately, 6.7 μL of 1.5 M Tris-HCl, pH 7.8 was added for neutralization. Elution was performed twice to obtain 200 μL of purified antibody.
Cation exchange chromatography (IEC) analysis of WT-IgG1, WT-IgG2, and WT-IgG4 Analysis by cation exchange chromatography to evaluate the homogeneity of purified WT-IgG1, WT-IgG2, and WT-IgG4 Went.
IEC Evaluation Method: System Waters Alliance
Column ProPac WCX-10 (Dionex)
Mobile phase A: 25 mM MES-NaOH, pH 6.1
B: 25mM MES-NaOH, 250mM Na-Acetate, pH6.1
Flow rate / wavelength 0.5ml / min, 280nm
Gradient B: 50% -75% (75min) WT-IgG1 analysis
B: 30% -55% (75 min) WT-IgG2 and WT-IgG4 analysis results are shown in FIG. WT-IgG1 and WT-IgG4 were single peaks by ion exchange analysis, but WT-IgG2 was found to have multiple peaks, and IgG2 molecules have heterogeneity compared to IgG1 and IgG4. I found that there were many. In fact, IgG2 isotypes have been reported to have heterogeneity derived from disulfide bonds in the hinge region (Chu GC, Chelius D, Xiao G, Khor HK, Coulibaly S, Bondarenko PV. Accumulation of Succinimide In a Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under Elevated Temperatures. Pharm Res. 2007 Mar 24; 24 (6): 1145-56), the IgG2 heteropeak shown in Fig. 22 is also derived from the target substance / related substance. it is conceivable that. It is difficult to manufacture a large amount as a drug while maintaining the heterogeneity of the target substance / related substance, and the antibody molecule developed as a drug is preferably as homogeneous as possible (low heterogeneity). You should have it. Therefore, it was considered that wild-type IgG2 has a problem in the uniformity that is important in developing an antibody as a pharmaceutical. In fact, in US20060194280 (A1), various types of heteropeaks derived from disulfide bonds were observed in natural IgG2 in ion exchange chromatography analysis, and it was also reported that biological activity differs between these peaks. In US20060194280 (A1), refolding in the purification process has been reported as a method for unifying this heteropeak, but it is costly and cumbersome to use these processes in production, and therefore, preferably amino acid substitution is used. Thus, a method for unifying hetero peaks is desirable. In order to develop it as a medicine, it was considered desirable to resolve the heterogeneity derived from the disulfide bond in the hinge region, but no method for solving this by amino acid substitution has been reported so far.

Preparation and Evaluation of Modified WT-IgG2 CH1 Domain and Hinge Region As shown in FIG. 23, various disulfide bond patterns can be considered for the IgG2 molecule. As a cause of heterogeneity derived from the hinge region of IgG2, it was considered that disulfide bonds were crossed and free cysteine was present. IgG2 has two cysteines in the upper hinge region (EU numbering 219 and 220, cysteines adjacent to the two cysteines in this upper hinge, EU numbering 131 cysteine and L in the CH1 domain of the H chain) Cysteine at the C-terminal of the chain and two cysteines in the same upper hinge of the dimerizing partner H chain, that is, around the upper hinge of IgG2, a total of 8 cysteines are adjacent when H2L2 is associated. This suggests that disulfide bond crossover and various heterogeneities due to free cysteine exist.
In order to reduce the heterogeneity derived from the hinge region of IgG2, the hinge region sequence and CH1 domain of IgG2 were modified. In IgG2, a study was conducted to avoid crossover of disulfide bonds and heterogeneity due to free cysteine. As a result of examination of various variants, among the wild type IgG2 constant region sequences, the EU numbering 131st cysteine and 133th arginine present in the CH1 domain of the H chain were changed to serine and lysine, respectively, and the H chain upper hinge By altering the 219th cysteine of the EU numbering 219 in Serine (hereinafter referred to as IgG2-SKSC) (IgG2-SKSC: SEQ ID NO: 120), heterogeneity can be avoided without reducing thermal stability It was considered possible. Thereby, it is considered that the covalent bond between the H chain and L chain of IgG2-SKSC is uniformly formed by disulfide bond between the EU numbering cysteine at position 220 and the cysteine at the C terminal of the L chain (FIG. 24).
Production, expression and purification of IgG2-SKSC expression vector were carried out by the method described in Example 1. In order to evaluate the homogeneity of purified IgG2-SKSC and wild type IgG2 (WT-IgG2), analysis by cation exchange chromatography was performed.
IEC Evaluation Method: System Waters Alliance
Column ProPac WCX-10 (Dionex)
Mobile phase A: 25 mM MES-NaOH, pH 5.6
B: 25mM MES-NaOH, 250mM Na-Acetate, pH5.6
Flow rate / wavelength 0.5ml / min, 280nm
Gradient B: 50% -100% (75min)
The results are shown in FIG. As described above, WT-IgG2 has a plurality of peaks, but IgG2-SKSC was found to elute as a single peak. Heterogeneity derived from the disulfide bond in the hinge region of IgG2 is achieved by introducing a modification of IgG2-SKSC that forms a single disulfide bond between the EU numbering cysteine at position 220 and the C-terminal cysteine of the L chain. It was shown that it can be avoided. Moreover, as a result of measuring the heat denaturation intermediate temperature of WT-IgG1, WT-IgG2 and IgG2-SKSC by the same method as in Example 5, WT-IgG2 is a Fab domain having a lower Tm value than WT-IgG1. Although a peak was observed, the peak was not observed in IgG2-SKSC. From this, it was found that IgG2-SKSC is superior in thermal stability as compared with WT-IgG2.
Wild-type IgG2 was considered to have a problem in the homogeneity that is important in developing an antibody as a pharmaceutical, but this problem can be solved by using IgG2-SKSC as the constant region sequence of IgG2. It became clear that it was extremely useful in developing IgG2 as a pharmaceutical. IgG2-SKSC was also found to be useful from the viewpoint of excellent thermal stability.

[Example 9] Improvement of C-terminal heterogeneity of IgG molecules
Expression vector construction of WT-IgG1 H chain C-terminal ΔGK antibody As a heterogeneity of C-terminal sequence of antibody, C-terminal amino acid deletion of lysine residue and C-terminal 2-amino acid glycine and lysine deletion C Amidation of the terminal amino group has been reported (Johnson KA, Paisley-Flango K, Tangarone BS, Porter TJ, Rouse JC. Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal Biochem. 2007 Jan 1; 360 (1): 75-83.), It is desirable that these heterogeneities do not exist when developing as pharmaceuticals. In fact, in TOCILIZUMAB, which is a humanized PM-1 antibody, its main component is a sequence in which the lysine at the C-terminal amino acid present on the base sequence is deleted by post-translational modification. It exists as a jenity. Therefore, the C-terminal amino acid was modified for the purpose of reducing heterogeneity of the C-terminal amino acid. Specifically, amidation of the C-terminal amino group by deletion of the glycine and lysine of the 2 amino acids at the C-terminal by previously deleting the lysine and glycine at the C-terminal of the heavy chain constant region of wild-type IgG1 on the base sequence We examined whether it was possible to suppress it.
Using the humanized PM1 antibody (WT) pB-CH vector obtained in Example 1, mutations were introduced into the H chain C-terminal sequence. Using QuikChange Site-Directed Mutagenesis Kit (Stratagene), a mutation that uses the nucleotide sequence encoding Lys at EU numbering 447 and / or Gly at EU numbering 446 as a stop codon by the method described in the attached manual Introduced. Thus, an antibody in which the C-terminal 1 amino acid lysine (EU numbering 447) has been deleted in advance, the C-terminal 2 amino acid glycine (EU numbering 446), and an antibody in which lysine (EU numbering 447) has been deleted in advance Was made. H chain C-terminal ΔK antibody and H chain C-terminal ΔGK antibody were obtained by expressing with humanized PM1 antibody L chain. Expression and purification were carried out by the method described in Example 1.
Cation exchange chromatography analysis of the purified H chain C-terminal ΔGK antibody was performed as follows. Using purified H chain C-terminal ΔGK antibody, analysis by cation exchange chromatography was performed by the following method to evaluate the influence of C-terminal deletion on heterogeneity. Cation exchange chromatography analysis conditions were as follows, and chromatograms of humanized PM1 antibody, H chain C-terminal ΔK antibody, and H chain C-terminal ΔGK antibody were compared.
Column: ProPac WCX-10, 4 x 250 mm (Dionex)
Mobile phase: A: 25 mmol / L MES / NaOH, pH 6.1
B: 25 mmol / L MES / NaOH, 250 mmol / L NaCl, pH 6.1
Flow rate: 0.5 mL / min
Gradient: 25% B (5 min) → (105 min) → 67% B → (1 min) → 100% B (5 min)
Detection: 280 nm
The analysis results of the unmodified humanized PM-1 antibody, the H chain C-terminal ΔK and the H chain C-terminal ΔGK antibody are shown in FIG. Non-patent literature (Chu GC, Chelius D, Xiao G, Khor HK, Coulibaly S, Bondarenko PV. Accumulation of Succinimide in a Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under Elevated Temperatures. Pharm Res. 2007 Mar 24; 24 (6): 1145-56), the basic peak that is later retained than the main peak contains the 449th Lys residue and the 447th Pro amide of the H chain C-terminal, but the base that was not found in the H chain C-terminal ΔK The H chain C-terminal ΔGK antibody showed a significant decrease in sex peaks, and it was found that the H chain C-terminal heterogeneity could be reduced only by deleting two amino acids at the H chain C terminal. .
In order to evaluate the influence of the deletion of 2 residues at the H chain C-terminal on the thermal stability, the thermal denaturation temperature of the H chain C-terminal ΔGK antibody was measured by DSC. For DSC measurement, the buffer was replaced by dialyzing against 20 mM acetate buffer containing 150 mM NaCl, pH 6.0. The humanized PM1 antibody, the H chain C-terminal ΔGK antibody and the reference solution (external dialysis solution) were sufficiently degassed, and then each was enclosed in a calorimeter cell, and heat equilibration at 40 ° C. was sufficiently performed. The DSC scan was then performed at 40 ° C to 100 ° C at a scan rate of about 1 K / min. The resulting denaturation peak was assigned with reference to non-patent literature (Rodolfo et al., Immunology Letters, 1999, p47-52), confirming that the C-terminal deletion does not affect the thermal denaturation temperature of the CH3 domain. did.
This makes it possible to reduce the heterogeneity of the C-terminal amino acid without affecting the thermal stability of the antibody by deleting the C-terminal lysine and glycine of the H chain constant region in advance in the base sequence. became. In the human antibody constant regions IgG1, IgG2, and IgG4, the C-terminal sequences are all EU numbering 447 is Lys and EU numbering 446 is Gly. It is considered that the method for reducing the tee is applicable to the IgG2 constant region and the IgG4 constant region, or variants thereof.

[Example 10] Preparation of a new optimized constant region M14ΔGK sequence In an antibody drug intended to neutralize an antigen, an effector function such as ADCC of the Fc region is not necessary, and therefore binding to the Fcγ receptor is not required. is necessary. In view of immunogenicity and side effects, binding to Fcγ receptor is considered undesirable (Strand V, Kimberly R, Isaacs JD. Biologic therapies in rheumatology: lessons learned, future directions. Nat Rev Drug Discov. 2007 Jan; 6 (1): 75-92., Gessner JE, Heiken H, Tamm A, Schmidt RE. The IgG Fc receptor family. Ann Hematol. 1998 Jun; 76 (6): 231-48. Humanized anti-IL-6 receptor IgG1 antibody TOCILIZUMAB specifically binds to IL-6 receptor and neutralizes its biological action to treat IL-6-related diseases such as rheumatoid arthritis And binding to the Fcγ receptor is unnecessary.

Preparation and evaluation of optimized constant region M14ΔGK for Fcγ receptor non-binding
As a method for reducing the binding to the Fcγ receptor, a method of changing the IgG antibody isotype from IgG1 to IgG2 or IgG4 isotype can be considered (Ann Hematol. 1998 Jun; 76 (6): 231-48.). As a method for completely eliminating the binding to the Fcγ receptor, a method for introducing an artificial modification into the Fc region has been reported. For example, since anti-CD3 and anti-CD4 antibodies cause side effects due to the effector functions of the antibodies, amino acid mutations (Cole MS, Anasetti C, Tso JY. Human IgG2 variants of chimeric anti-CD3 are nonmitogenic to T cells.J Immunol. 1997 Oct 1; 159 (7): 3613-21., Reddy MP, Kinney CA, Chaikin MA, Payne A, Fishman-Lobell J, Tsui P, Dal Monte PR, Doyle ML, Brigham-Burke MR, Anderson D, Reff M, Newman R, Hanna N, Sweet RW, Truneh A. Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4. J Immunol. 2000 Feb 15; 164 (4): 1925-33.) Fcγ receptor non-binding anti-CD3 and anti-CD4 antibodies have been clinically tested (Strand V, Kimberly R, Isaacs JD. Biologic therapies in rheumatology: lessons learned, future directions. Nat Rev Drug Discov. 2007 Jan; 6 (1): 75-92., Chau LA, Tso JY, Melrose J, Madrenas J. HuM291 (Nuvion), a humanized Fc receptor-nonbinding antibody against CD3, anergizes peripheral blood T cells as partial agonist of the T cell receptor. Transplantation. 2001 Apr 15; 71 (7): 941-50.). In addition, IgG1 FcγR binding sites (EU numbering: 233, 234, 235, 236, 327, 330, 331) are IgG2 (EU numbering: 233, 234, 235, 236) and IgG4 (EU numbering: 327, 330, It has been reported that Fcγ receptor non-binding antibody can be produced by using the 331st sequence (Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol. Cells. 2005 Aug 31; 20 (1): 17-29. Review.). However, when all of these mutations are introduced into IgG1, a new peptide sequence of 9 amino acids that can become a non-naturally occurring T-cell epitope peptide appears, increasing the risk of immunogenicity. In developing as a medicine, it is desirable that the risk of immunogenicity is as low as possible.
In order to solve the above-mentioned problems, modification of IgG2 to the constant region was examined. The constant region of IgG2 is EU numbering: 233, 234, 235, 236 among FcγR binding sites, but EU numbering: 327, 330, and 331th among FcγR binding sites are non-binding IgG4. Since it is a different sequence, it is necessary to modify EU numbering: amino acids 327, 330 and 331 to the sequence of IgG4 (G2Δa in Eur J Immunol. 1999 Aug; 29 (8): 2613-24). However, IgG4 is EU numbering: 339th amino acid is alanine, whereas IgG2 is threonine. EU numbering: 327, 330, 331 amino acids are naturally changed to IgG4 sequence. A new peptide sequence of 9 amino acids that can be a non-existing T-cell epitope peptide appears, which increases the risk of immunogenicity. Thus, in addition to the above-mentioned modification, it was found that IgG2 EU numbering: it is possible to prevent the appearance of a new peptide sequence by modifying the 339th threonine to alanine.
In addition to these mutations, the mutation from the 397th methionine to valine in the EU numbering of IgG2 that improves the stability of IgG2 found in Example 7 under acidic conditions, the hinge region found in Example 8 In order to improve the heterogeneity derived from the disulfide bond, mutations in the 131st cysteine to serine, 133th arginine to lysine, and 219th cysteine to serine were introduced. Furthermore, with the introduction of the 131st and 133rd mutations, a new 9-amino acid peptide sequence that can become a non-naturally occurring T-cell epitope peptide appears, resulting in an immunogenicity risk. Therefore, the EU numbering 137th glutamic acid is introduced. By introducing a mutation from glycine to glycine and a mutation from serine to glycine at position 138, the peptide sequence from the 131st position to the 139th position was made identical to the naturally occurring human sequence. Furthermore, in order to reduce heterogeneity derived from the C terminus, the glycine and lysine at positions 446 and 447 of the EU numbering of the H chain C terminus were deleted. The constant region sequence into which all of these mutations were introduced was designated as M14ΔGK (M14ΔGK: SEQ ID NO: 24). M14ΔGK is a 9 amino acid new peptide sequence that can be a T-cell epitope peptide. There is one site where a mutation from cysteine to serine is introduced at position 219, but cysteine and serine are similar in amino acid sequence. The risk of immunogenicity is considered to be extremely small, and no change in immunogenicity was observed in the immunogenicity prediction by TEPITOPE.
An expression vector of an H chain antibody sequence (M14ΔGK: SEQ ID NO: 24, WT-M14ΔGK: SEQ ID NO: 113) having WT as a variable region sequence and M14ΔGK as a constant region sequence was obtained by the method described in Example 1. This was expressed and purified by the method described in Example 1 using WT-M14ΔGK as the H chain and WT as the L chain.
In the same manner, mutations were introduced into EU numbering: 233, 234, 235, 236, 327, 330, 331, and 339 in order to reduce binding to the Fcγ receptor in the IgG1 constant region (Eur J Immunol. 1999 Aug; 29 (8): 2613-24, G1Δab), and EU numbering 446th and 447th were deleted to reduce C-terminal heterogeneity (Example 9) WT-M17ΔGK (M17ΔGK: sequence) No. 116, WT-M17ΔGK: SEQ ID NO: 115). In order to reduce binding to the Fcγ receptor in the IgG4 constant region, mutations were introduced at positions 233, 234, 235 and 236 (Eur J Immunol. 1999 Aug; 29 (8): 2613-24, G4Δb, In order to reduce the immunogenicity risk, new non-human sequences are generated in the modification, and in order to reduce the immunogenicity risk, in addition to the above modifications, the numbering of the EU to make the disulfide bond mode of the hinge region the same as M14ΔGK : Mutations are introduced at the 131st, 133th, 137th, 138th, 214th, 217th, 219th, 220th, 221th, 222nd, and the mutation is introduced at the 409th EU numbering in order to improve the stability under acidic conditions ( Example 7), EU numbering 446th and 447th were deleted in order to reduce C-terminal heterogeneity (Example 9) WT-M11ΔGK (M11ΔGK: sequence) No.: 25, WT-M11ΔGK: SEQ ID NO: 114) were prepared expression vector. It was expressed and purified by the method described in Example 1 using WT-M17ΔGK or WT-M11ΔGK as the H chain and WT as the L chain.

Evaluation of Fcγ receptor binding activity of WT-M14ΔGK, WT-M17ΔGK, WT-M11ΔGK
Evaluation of binding to FcγRI was performed as follows. Using Biacore T100, human-derived Fcγ receptor I (hereinafter FcγRI) immobilized on the sensor chip interacts with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M1ΔGK7 used as analytes, Compared. Recombinant Human FcRIA / CD64 (R & D systems) was used as human-derived FcγRI, and IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK were used as samples. FcγRI was immobilized on sensor chip CM5 (BIACORE) by the amine coupling method. The final fixed amount of hFcγRI was about 13000 RU (resonance units). HBS-EP + was used as a running buffer, and the flow rate was 20 μL / min. Samples were adjusted to a concentration of 100 μg / mL using HBS-EP +. The analysis was performed by injecting 10 μL of the antibody solution for 2 minutes as a binding phase, and then switching to HBS-EP + for a 4-minute dissociation phase. After completion of the dissociation phase, the sensor chip was regenerated by injecting 20 μL of 5 mM sodium hydroxide. This binding / dissociation / regeneration was regarded as one cycle of analysis, and various antibody solutions were injected to obtain sensorgrams. The analyte was run in the order of IgG4, IgG2, IgG1, M11, M14, and M17, and this was repeated twice. The result of comparison of the measured binding amount data is shown in FIG. As a result, the amount of binding decreased in the order of IgG1> IgG4 >> IgG2 = M11ΔGK = M14 ΔGK = M17ΔGK, and wild-type IgG2, M11ΔGK, M14ΔGK, and M17ΔGK bound to FcγRI rather than wild-type IgG1 and IgG4. Was found to be weak.
Evaluation of binding to FcγRIIa was performed as follows. Using Biacore T100, human-derived Fcγ receptor IIa (hereinafter FcγRIIa) immobilized on a sensor chip interacts with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK used as analytes, and the amount of binding is compared. did. As human-derived FcγRIIa, Recombinant Human FcRIIA / CD32a (R & D systems) was used, and IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK were used as samples. FcγRIIa was immobilized on sensor chip CM5 (BIACORE) by the amine coupling method. Finally, about 3300 RU of FcγRIIa was immobilized. HBS-EP + was used as a running buffer, and the flow rate was 20 μL / min. Thereafter, the running buffer was passed until the baseline became stable, and the measurement was performed after the baseline was stabilized. The IgG isotype (IgG1, IgG2, IgG4) and the antibody (M11ΔGK, M14ΔGK, M17ΔGK) into which each IgG isotype was introduced as an analyte interacted with the immobilized FcγRIIa, and the amount of binding was observed. HBS-EP + was used as the running buffer, the flow rate was 20 μL / min, and the measurement temperature was 25 ° C. Each IgG and variant was adjusted to 100 μg / mL, and 20 μL was flowed as an analyte to interact with the immobilized FcγRIIa. After the interaction, the analyte was dissociated from FcγRIIa by flowing 200 μL of running buffer, and the sensor chip was regenerated. The analytes were flowed in the order of IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK, and this was repeated twice. The result of comparing the measured binding amount data is shown in FIG. As a result, the amount of binding decreased in the order of IgG1> IgG2 = IgG4> M11ΔGK = M14ΔGK = M17ΔGK. Became clear.
Evaluation of binding to FcγRIIb was performed as follows. Using Biacore T100, human-derived Fcγ receptor IIb (hereinafter referred to as FcγRIIb) immobilized on a sensor chip interacts with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, M17ΔGK used as analytes, and the amount of binding is compared. did. Recombinant Human FcRIIB / C (R & D systems) was used as human-derived FcγRIIb, and measurement was performed using IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK as samples. FcγRIIb was immobilized on sensor chip CM5 (BIACORE) by the amine coupling method. Finally, about 4300 RU of FcγRIIb was immobilized. Thereafter, the running buffer was passed until the baseline became stable, and the measurement was performed after the baseline was stabilized. The IgG isotype (IgG1, IgG2, IgG4) and the antibody (M11ΔGK, M14ΔGK, M17ΔGK) into which each IgG isotype was introduced as an analyte interacted with the immobilized FcγRIIb, and the amount of binding was observed. The running buffer was HBS-EP + (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20), the flow rate was 20 μL / min, and the measurement temperature was 25 ° C. Each IgG and variant was adjusted to 200 μg / mL, and 20 μL was flowed as an analyte to interact with the immobilized FcγRIIb. After the interaction, the analyte was dissociated from FcγRIIb by flowing 200 μL of running buffer, and the sensor chip was regenerated. The analytes were flowed in the order of IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK, and this was repeated twice. The result of comparing the measured binding amount data is shown in FIG. As a result, the amount of binding decreased in the order of IgG4>IgG1>IgG2> M11ΔGK = M14ΔGK = M17ΔGK. Became clear.
Evaluation of binding to FcγRIIIa was performed as follows. Using Biacore T100, human-derived Fcγ receptor IIIa (hereinafter referred to as FcγRIIIa) immobilized on a sensor chip interacts with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, M17ΔGK used as analytes, and the amount of binding is compared. did. As human-derived FcγRIIIa, hFcγRIIIaV-His6 (recombinant hFcγRIIIaV-His6: in-house preparation) was used, and IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK were used as samples. FcγRIIIa was immobilized on sensor chip CM5 (BIACORE) by the amine coupling method. The final fixed amount of hFcγRIIIaV-His6 was about 8200 RU (resonance units). HBS-EP + was used as a running buffer, and the flow rate was 5 μL / min. Samples were prepared with HBS-EP + to a concentration of 250 μg / mL. The analysis was performed by injecting 10 μL of the antibody solution for 2 minutes as a binding phase, and then switching to HBS-EP + for a 4-minute dissociation phase. After completion of the dissociation phase, the sensor chip was regenerated by injecting 20 μL of 5 mM hydrochloric acid. This binding / dissociation / regeneration was regarded as one cycle of analysis, and various antibody solutions were injected to obtain sensorgrams. The analytes were run in the order of IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK. The result of comparing the measured binding amount data is shown in FIG. As a result, the amount of binding decreased in the order of IgG1 >>IgG4>IgG2>M17ΔGK> M11ΔGK = M14ΔGK, and M11ΔGK, M14ΔGK, and M17ΔGK may have weaker binding to FcγRIIIa than wild type IgG1, IgG2, and IgG4 It became clear. In addition, it was revealed that M11ΔGK and M14ΔGK are weaker bindings compared to M17ΔGK containing the G1Δab mutation reported in Eur J Immunol. 1999 Aug; 29 (8): 2613-24.
From the above, it was confirmed that the binding of WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK to various Fcγ receptors was significantly reduced compared to wild-type IgG1. By using WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK as constant regions, avoiding the immunogenicity risk derived from incorporation into APC via Fcγ receptor and side effects derived from effector functions such as ADCC And neutralizing the antigen is useful as a constant region sequence of the target antibody drug.

High concentration stability test of WT-M14ΔGK, WT-M17ΔGK, WT-M11ΔGK
The stability of high-concentration preparations of WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK was evaluated. Dialyze (EasySEP, TOMY) the purified antibodies of WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, WT-M11ΔGK against a solution of 20 mM histidine chloride, 150 mM NaCl, pH 6.5, and then concentrate with an ultrafiltration membrane Then, a high concentration stability test was conducted. The conditions are as follows.
Antibodies: WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, WT-M11ΔGK
Buffer: 20 mM histidine chloride, 150 mM NaCl, pH 6.5
Concentration: 61 mg / mL
Storage temperature and duration: 40 ℃ -2W, 40 ℃ -1M, 40 ℃ -2M
Aggregate Evaluation Method: System Waters Alliance
Column G3000SWxl (TOSOH)
Mobile phase 50 mM sodium phosphate, 300 mM KCl, pH 7.0
Flow velocity / wavelength 0.5ml / min, 220nm
Dilute sample to 1/100 for analysis
The aggregate content of the formulation after storage at initial (immediately after preparation of the formulation) and after storage under each condition was evaluated by the gel filtration chromatography described above, and the amount of change in aggregate content from the initial is shown in FIG. As a result, the increased amount of aggregates of WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK was lower than that of WT-IgG1, and was about ½ of the increased amount of WT aggregates. As shown in FIG. 32, WT-IgG1 and WT-M17ΔGK were about the same in terms of the amount of Fab fragment increase, but WT-M14ΔGK and WT-M11ΔGK were about 1/4 of the WT Fab fragment increase. Met. As described in WO 2003/039485, the degradation pathway of an IgG type antibody preparation mainly includes the production of aggregates and the production of Fab degradation products. WT-M14ΔGK and WT-M11ΔGK were found to be superior in formulation stability in two respects to the production of aggregates and Fab fragments compared to WT-IgG1. As a result, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK may be used as the constant region even for antibodies for which high-concentration solution preparations that can be developed as pharmaceuticals were not sufficiently stable in the IgG1 constant region. It was considered that a high-concentration solution preparation having high stability could be prepared.
In particular, M14ΔGK improves the instability under acidic conditions inherent in IgG2 molecules, improves heterogeneity derived from the disulfide bond in the hinge region, does not bind to Fcγ receptor, and can be a T-cell epitope peptide 9 It was considered to be extremely useful as a novel constant region sequence that minimizes the new peptide sequence of amino acids and is superior to IgG1 in stability in high concentration preparations.

[Example 11] Preparation of PF1-M14ΔGK antibody The variable region of PF1 (constant region is IgG1) prepared in Example 5 was excised with XhoI / NheI, and the constant of M14ΔGK (variable region was WT) prepared in Example 7 The region was excised with NheI / NotI, and the two H-chain antibody gene fragments were inserted into an animal cell expression vector to prepare a target PF1-M14ΔGK H-chain expression vector (PF1_H-M14ΔGK: SEQ ID NO: 117). The PF1-M14ΔGK antibody was expressed and purified by the method described in Example 1 using PF1_L as the L chain.
The PF1-M14ΔGK antibody was superior to WT (humanized PM-1 antibody) in various respects as a pharmaceutical agent for anti-IL-6 receptor antibody, and was thought to be extremely useful.

[Example 12] Production and evaluation of M31ΔGK For M14ΔGK produced in Example 10, EU numbering: M31ΔGK in which the 330th, 331th and 339th positions were modified to the IgG2 sequence was prepared (M31ΔGK: SEQ ID NO: 118). An expression vector of an H chain antibody sequence (WT-M31ΔGK: SEQ ID NO: 119) having WT as a variable region sequence and M31ΔGK as a constant region sequence was prepared by the method described in Example 1, and WT was used as an H chain. WT-M31 was expressed and purified by the method described in Example 1 using -M31ΔGK and WT as the L chain.
In addition to WT-M31, cation-exchange chromatography analysis of WT-IgG2 and WT-M14ΔGK simultaneously expressed and purified was performed as follows. The cation exchange chromatography analysis conditions were as follows, and the chromatograms of WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK were compared.
Column: ProPac WCX-10, 4 x 250 mm (Dionex)
Mobile phase: A: 25 mmol / L MES / NaOH, pH 6.1
B: 25 mmol / L MES / NaOH, 250 mmol / L NaCl, pH 6.1
Flow rate: 0.5 mL / min
Gradient: 0% B (5 min) → (65 min) → 100% B → (1 min)
Detection: 280 nm
The analysis results of WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK are shown in FIG. Although WT-IgG2 has a plurality of peaks, WT-M31ΔGK was found to elute as a single peak, like WT-M14ΔGK. It was shown that heterogeneity derived from the disulfide bond in the hinge region of IgG2 can be avoided also in WT-M31ΔGK.

PF1_H-IgG1に対し、Kabatナンバリング66番から94番を上記の候補配列に置換することにより、H96-IgG1（配列番号：１３４アミノ酸配列））を設計した。抗体可変領域については、合成オリゴDNAを組み合わせたPCR法(assembly PCR)により作製した。定常領域については、IgG1の発現ベクターからPCR法により増幅した。Assembly PCR法により抗体可変領域と定常領域を結合させ、動物細胞発現用ベクターへ挿入した。H96/PF1L-IgG1の発現、精製を実施例１に記載した方法により行なった。 [Example 13] Production of fully humanized antibody F2H / L39-IgG1
Completely humanization of framework sequence of PF1 antibody PF1_H prepared in Example 5 contains only arginine of No. 71 (Kabat numbering, Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH). This is not preferable from the viewpoint of immunogenicity. In general, the 71st residue of the H chain is an important sequence that determines the structure of HCDR2, and it has been reported that it is important for the binding activity of the mouse PM1 antibody when actually producing a humanized PM1 antibody. It has been clarified that substitution of arginine with valine significantly reduces the binding activity (Cancer Research 53, 851-856, 1993). Similarly, PF1_H is classified into the VH4 family of human germline genes, but No. 71 in the VH4 family is highly conserved as valine. It was done.
Therefore, in order to completely remove the mouse sequence while leaving No. 71 as the arginine residue, the human germline gene and the sequence of the reported human antibody were investigated. We searched for conserved sequences that are important for the maintenance of. As a result, candidate sequences in which important residues were conserved although the homology with PF1_H shown in Table 9 was low were found.

H96-IgG1 (SEQ ID NO: 134 amino acid sequence)) was designed by substituting Kabat numbering Nos. 66 to 94 with the above candidate sequences for PF1_H-IgG1. The antibody variable region was prepared by a PCR method (assembly PCR) in which synthetic oligo DNAs were combined. The constant region was amplified from an IgG1 expression vector by PCR. The antibody variable region and the constant region were combined by Assembly PCR method and inserted into an animal cell expression vector. H96 / PF1L-IgG1 was expressed and purified by the method described in Example 1.

以上より、PF1抗体のH鎖をH96にすることで、Tm値およびアフィニティーを維持したままで、PF1抗体に残されていたマウス配列を完全に除去し、PF1抗体のフレームワークを完全ヒト化した抗体が得られた。H96/PF1L-IgG1は、フレームワーク配列にマウス由来配列が存在しないことから、免疫原性の観点で特に優れていると考えられた。 Evaluation of framework fully humanized antibody H96 / PF1L-IgG1 Using purified H96 / PF1L-IgG1, the Tm value was measured by the method described in Example 5. In principle, the affinity was measured under the same conditions as in Example 5. However, the concentration of SR344 was adjusted to 0, 0.36, and 1.4 μg / mL, and the dissociation phase was measured for 15 minutes.
As a result, H96 / PF1L-IgG1 showed almost the same Tm value and affinity as PF1-IgG1 (Table 10).

From the above, by setting the H chain of the PF1 antibody to H96, the mouse sequence remaining in the PF1 antibody was completely removed while maintaining the Tm value and affinity, and the PF1 antibody framework was fully humanized. An antibody was obtained. H96 / PF1L-IgG1 was thought to be particularly excellent from the viewpoint of immunogenicity because there was no mouse-derived sequence in the framework sequence.

Production of F2H / L39-IgG1 with reduced pI and reduced immunogenicity risk As shown in Example 4, it is possible to improve plasma retention by altering the amino acid of the variable region of the antibody to lower pI. It is clear. Therefore, the following amino acid substitutions were further introduced into H96-IgG1 prepared above. In order to reduce pI, substitution of lysine No. 64 with glutamine and substitution of glycine No. 65 with aspartic acid were introduced. In addition, in order to reduce the immunogenicity risk, substitution of glutamine No. 105 with glutamine and substitution of threonine No. 107 with isoleucine were introduced. Furthermore, substitution of valine No. 95 and leucine No. 95 and substitution of isoleucine No. 99 to alanine, which are modification of affinity enhancement obtained in Example 2, were introduced. F2H-IgG1 (SEQ ID NO: 135 (amino acid sequence)) in which these amino acid substitutions were introduced into H96-IgG1 was prepared by the method described in Example 1.
In addition, the following amino acid substitutions were introduced into PF1L. In order to reduce pI, substitution of glutamine No. 27 with glutamic acid and substitution of 55-leucine with glutamic acid were introduced. L39 (SEQ ID NO: 136 (amino acid sequence)) in which these amino acid substitutions were introduced into PF1L was prepared by the method described in Example 1. F2H / L39-IgG1 was expressed and purified by the method described in Example 1.

その結果、F2H/L39-IgG1は、KD値としては10^-11オーダーを維持しており、極めて強いアフィニティーを有しているが、k_aがPF1-IgG1と比較して1/2程度に低下していることが分かった。 Affinity analysis of F2H / L39-IgG1 for human IL-6 receptor by Biacore Affinity measurement of humanized PM1 antibody (wild type, WT), PF1 antibody (made in Example 5) and F2H / L39-IgG1 was performed. . In principle, this measurement was performed under the same conditions as in Example 4. However, the concentration of SR344 was adjusted to 0, 0.36, and 1.4 μg / mL, and the dissociation phase was measured for 15 minutes (Table 11).

Lowering a result, F2H / L39-IgG1, as the KD value maintains a 10 ^-11 order has the very strong affinity, compared k _a is the PF1-IgG1 about 1/2 I found out that

Evaluation of human IL-6 receptor neutralizing activity of F2H / L39-IgG1 The neutralizing activity of humanized PM1 antibody (wild type, WT) and F2H / L39-IgG1 was evaluated by the method shown in Example 1. However, human interleukin-6 (TORAY) concentration was 600 ng / mL, and neutralization activity was evaluated. As shown in FIG. 34, it was revealed that F2H / L39-IgG1 has an extremely strong activity of 100 times or more as a 100% inhibitory concentration compared with WT.

Isoelectric point evaluation of F2H / L39-IgG1 by isoelectric focusing
The isoelectric point of F2H / L39-IgG1 was measured by the method described in Example 3. The isoelectric point of F2H / L39-IgG1 was 5.5, and it was considered that the retention in plasma was further improved by further decreasing the isoelectric point as compared with the PF1 antibody prepared in Example 5.
The theoretical isoelectric point of the variable region (VH, VL arrangement) of F2H / L39 was calculated by GENETYX (GENETYX CORPORATION), and the theoretical isoelectric point was 4.3. Since the theoretical isoelectric point of WT was 9.20, F2H / L39 was obtained in which the theoretical isoelectric point of the variable region was reduced by about 4.9 by amino acid substitution from WT.

PK / PD test by cynomolgus monkeys with F2H / L39-IgG1 Pharmacokinetics (PK) and drug efficacy (PD) in cynomolgus monkeys of humanized PM1 antibody (wild type, WT), PF1 antibody and F2H / L39-IgG1 were evaluated. WT, PF1 and F2H / L39-IgG1 were subcutaneously administered at a single dose of 1.0 mg / kg, and blood was collected before and over time. In the same manner as in Example 6, the plasma concentration of each antibody was measured. FIG. 35 shows changes in plasma concentrations of WT, PF1, and F2H / L39-IgG1. In order to evaluate the degree of neutralization of cynomolgus monkey membrane IL-6 receptor, cynomolgus monkey IL-6 5 μg / kg was administered from the 3rd day (day 3) to 10th day (day 10) after antibody administration. Was administered subcutaneously every day, and the CRP concentration of each individual was measured 24 hours later. FIG. 36 shows changes in CRP concentration during administration of WT and F2H / L39. In order to evaluate the degree of neutralization of the cynomolgus monkey soluble IL-6 receptor, the concentration of unbound cynomolgus monkey soluble IL-6 receptor in cynomolgus monkey plasma was measured. FIG. 37 shows changes in the concentration of unbound cynomolgus monkey soluble IL-6 receptor upon administration of WT and F2H / L39.
From these results, WT and PF1 showed almost the same plasma concentration transition, whereas F2H / L39-IgG1 with a lower pI maintained a higher antibody plasma concentration than these. In addition, F2H / L39-IgG1, which has a strong affinity for IL-6 receptor, was found to maintain a lower CRP concentration and non-binding cynomolgus monkey soluble IL-6 receptor concentration than WT. It was.

[Example 14] Evaluation of plasma retention of WT-M14
Method for predicting plasma retention in humans
The reason why IgG molecules have a long plasma retention (slow disappearance) is because FcRn known as a salvage receptor for IgG molecules is functioning (Nat Rev Immunol. 2007 Sep; 7 (9): 715 -twenty five). IgG molecules taken into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions in the endosome (around pH 6.0). IgG molecules that could not bind to FcRn go to lysosomes and are degraded by lysosomes, but IgG molecules bound to FcRn migrate to the cell surface and dissociate from FcRn under neutral conditions in plasma (around pH 7.4) Return to the plasma again.
IgG1, IgG2, IgG3, and IgG4 isotypes are known as IgG type antibodies. The plasma half-life in these humans is about 36 days for IgG1, IgG2, about 29 days for IgG3, and 16 for IgG4. (Nat Biotechnol. 2007 Dec; 25 (12): 1369-72.), And IgG1 and IgG2 are considered to have the longest retention in plasma. In general, antibody isotypes are IgG1, IgG2, and IgG4. However, as a method for further improving the retention of these IgG antibodies in plasma, the above-mentioned human FcRn binding activity can be achieved by modifying the sequence of the constant region of IgG. Has been reported (J Biol Chem. 2007 Jan 19; 282 (3): 1709-17, J Immunol. 2006 Jan 1; 176 (1): 346-56).
Because there are species differences between mouse FcRn and human FcRn (Proc Natl Acad Sci US A. 2006 Dec 5; 103 (49): 18709-14), the retention of IgG antibodies with modified constant region sequences in plasma To predict human FcRn binding and plasma retention in human FcRn transgenic mice (Int Immunol. 2006 Dec; 18 (12): 1759-69) .

Evaluation of binding to human FcRn
FcRn is a complex of FcRn and β2-microglobulin. Oligo DNA primers were prepared based on the published human FcRn gene sequence (J. Exp. Med. 180 (6), 2377-2381 (1994)). A human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) was used as a template, and a DNA fragment encoding the full length of the gene was prepared by PCR using the prepared primer. Using the obtained DNA fragment as a template, a DNA fragment encoding an extracellular region (Met1-Leu290) including a signal region was amplified by PCR and inserted into an animal cell expression vector (human FcRn amino acid sequence SEQ ID NO: 140) . Similarly, oligo DNA primers were prepared based on the published human β2-microglobulin gene sequence (Proc. Natl. Acad. Sci. USA 99 (26), 16899-16903 (2002)). A human cDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH) was used as a template, and a DNA fragment encoding the full length of the gene was prepared by PCR using the prepared primer. Using the obtained DNA fragment as a template, the DNA fragment encoding β2-microglobulin full length (Met1-Met119) including the signal region was amplified by PCR and inserted into an animal cell expression vector (human β2-microglobulin amino acid sequence SEQ ID NO: : 141).
The soluble human FcRn was expressed by the following procedure. The prepared human FcRn and human β2-microglobulin plasmids were introduced into human embryonic kidney cancer cell-derived HEK293H strain (Invitrogen) by the lipofection method using 10% Fetal Bovine Serum (Invitrogen). The obtained culture supernatant was collected and purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences) according to the method of (J Immunol. 2002 Nov 1; 169 (9): 5171-80.). Thereafter, purification was performed with HiTrap Q HP (GE Healthcare).
Biacore 3000 was used for evaluation of binding to human FcRn, and human FcRn was interacted with human FcRn as an analyte to an antibody bound to Protein L or rabbit anti-human IgG Kappa chain antibody immobilized on a sensor chip. The affinity (KD) was calculated from the amount of binding. Specifically, Protein L or rabbit anti-human IgG Kappa chain antibody was immobilized on sensor chip CM5 (BIACORE) by amine coupling method using 50 mM Na-phosphate buffer containing 150 mM NaCl, pH 6.0 as running buffer. . Thereafter, the antibody was diluted with a running buffer containing 0.02% Tween20 and injected to bind the antibody to the chip. Then, human FcRn was injected, and the binding activity [0] of human FcRn to the antibody was evaluated.
Affinity was calculated using software, BIAevaluation. From the obtained sensorgram, the amount of hFcRn binding to the antibody just before the end of human FcRn injection was determined, and this was fitted by the steady state affinity method to calculate the affinity of the antibody against human FcRn.

Evaluation of plasma retention in human FcRn transgenic mice Evaluation of pharmacokinetics in human FcRn transgenic mice (B6.mFcRn-/-. HFcRn Tg line 276 + / + mice, Jackson Laboratories) was performed as follows. The antibody was administered intravenously at a dose of 1 mg / kg to mice and blood was collected in a timely manner. The collected blood was immediately centrifuged at 4 ° C. and 15,000 rpm for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set to −20 ° C. or lower until measurement was performed. Plasma concentration was measured using the ELISA method.

しかしながら、WT-IgG1とWT-M14のヒトFcRnトランスジェニックマウスにおける血漿中滞留性の評価を行った結果、図３８に示すとおり、WT-IgG1とWT-M14は同等の血漿中滞留性を示したことから、M14の定常領域はヒトにおいてもIgG1の定常領域と同等の血漿中滞留性を示すと考えられた。 Predictive evaluation of plasma retention in humans with WT-M14
As a result of evaluating the binding activity of WT-IgG1 and WT-M14 to human FcRn by BIAcore, as shown in Table 12, the binding activity of WT-M14 was slightly superior to WT-IgG1.

However, as a result of evaluating the plasma retention in human FcRn transgenic mice of WT-IgG1 and WT-M14, as shown in FIG. 38, WT-IgG1 and WT-M14 showed equivalent plasma retention Therefore, it was considered that the constant region of M14 exhibits the same plasma retention as the constant region of IgG1 in humans.

[Example 15] Production of WT-M44, WT-M58, and WT-M73 with improved plasma retention
Production of WT-M58 molecule As shown in Example 14, the retention of WT-M14 in plasma in human FcRn transgenic mice was equivalent to that of WT-IgG1. Known methods for improving plasma retention are methods that reduce the isoelectric point of antibodies and methods that enhance FcRn binding activity. The purpose is to improve plasma retention of WT-M14. The following modifications were introduced. Specifically, the EU numbering of WT-M31ΔGK prepared from WT-M14 in Example 4 was modified by changing the 397th valine to methionine, the 268th histidine to glutamine, and the 355th arginine to glutamine. No. glutamine was modified to glutamic acid. These four modifications were introduced into WT-M31ΔGK to prepare WT-M58 (SEQ ID NO: 142 (amino acid sequence)). The expression vector was prepared by the method of Example 1. The expression and purification of WT-M58 using WT-M58 as the H chain and L (WT) as the L chain were the methods described in Example 1. I went there.

Production of WT-M73 molecule On the other hand, WT-M44 (SEQ ID NO: 143 (amino acid sequence)) in which IgG1 was substituted for EU numbering: 434th was produced. Furthermore, WT-M83 (SEQ ID NO: 185 (amino acid sequence)) in which the 446th glycine and the 447th lysine were deleted was prepared in order to reduce the heterogeneity of the H chain C terminal relative to M44. In addition, WT-M73 (SEQ ID NO: 144 (amino acid sequence)) in which 434th EU numbering was substituted with alanine was prepared for WT-M58.
These expression vectors were prepared by the method of Example 1, using WT-M44, WT-M58 or WT-M73 as the H chain, and WT-M44 and WT using L (WT) as the L chain. -M58 and WT-M73 were expressed and purified by the method described in Example 1.

WT-IgG1、WT-M44、およびWT-M58のヒトFcRnトランスジェニックマウスにおける血漿中滞留性の評価を行った結果、図３９に示すとおり、WT-M58はWT-IgG1およびWT-M44と比較して血漿中滞留性の向上が確認された。さらに、WT-IgG1、WT-M44、WT-M58およびWT-M73のヒトFcRnトランスジェニックマウスにおける血漿中滞留性の評価を行った結果、図４０に示すとおり、WT-M44、WT-M58およびWT-M73はいずれもWT-IgG1と比較して血漿中滞留性の改善が確認され、その血漿中滞留性の改善効果はヒトFcRnへの結合能と相関した。なかでもWT-M73に関しては、WT-IgG1と比較して２８日後の血漿中濃度が約１６倍改善していたことから、ヒトにおいてもM73の定常領域を有する抗体はIgG1の定常領域を有する抗体と比較して大幅に血漿中滞留性が向上すると考えられた。 Predictive evaluation of plasma retention of WT-M44, WT-M58, and WT-M73 in humans
As a result of evaluating the binding activity of WT-IgG1, WT-M44, WT-M58 and WT-M73 to human FcRn by BIAcore, as shown in Table 13, the binding of WT-M44, WT-M58 and WT-M73 The activity was about 2.7 times, about 1.4 times and about 3.8 times better than WT-IgG1, respectively.

As a result of evaluation of plasma retention in human FcRn transgenic mice of WT-IgG1, WT-M44, and WT-M58, as shown in FIG. 39, WT-M58 was compared with WT-IgG1 and WT-M44. As a result, an improvement in plasma retention was confirmed. Furthermore, as a result of evaluating the retention in plasma in human FcRn transgenic mice of WT-IgG1, WT-M44, WT-M58 and WT-M73, as shown in FIG. 40, WT-M44, WT-M58 and WT -M73 was confirmed to have improved plasma retention compared to WT-IgG1, and the improvement in plasma retention correlated with the ability to bind to human FcRn. In particular, with regard to WT-M73, the plasma concentration after 28 days was improved by about 16 times compared with WT-IgG1, so that an antibody having a constant region of M73 is an antibody having a constant region of IgG1 even in humans. It was considered that the retention in plasma was significantly improved as compared with the above.

[Example 16] Effect of reducing heterogeneity by novel constant regions M14 and M58 in various antibodies As shown in Example 8, in humanized PM1 antibody (WT), which is an anti-IL-6 receptor antibody, the constant region was changed from IgG2. It was confirmed that the heterogeneity derived from the hinge region of IgG2 can be reduced by converting to M14. Therefore, we examined whether heterogeneity could be reduced by converting the constant region to M14 or M58 for IgG2 type antibodies other than humanized PM1 antibody.
As an antibody other than humanized PM1 antibody, anti-IL-6 receptor antibody F2H / L39 (F2H / L39_VH amino acid sequence SEQ ID NO: 145, F2H / L39 VL amino acid sequence SEQ ID NO: 146), anti-IL-31 receptor antibody A certain H0L0 (H0L0_VH amino acid sequence SEQ ID NO: 147, H0L0_VL amino acid sequence SEQ ID NO: 148) and anti-RANKL antibody DNS (DNS_VH amino acid sequence SEQ ID NO: 149, DNS_VL amino acid sequence SEQ ID NO: 150) were used. For each antibody, IgG1 (SEQ ID NO: 19), IgG2 (SEQ ID NO: 20), and M14 (SEQ ID NO: 24) or M58 (SEQ ID NO: 151) were prepared.
As an evaluation method of heterogeneity, evaluation by cation exchange chromatography was performed. The heterogeneity of the prepared antibody was evaluated using ProPac WCX-10 (Dionex) as the column, 20 mM Sodium Acetate, pH 5.0 as mobile phase A, and 20 mM Sodium Acetate, 1M NaCl, pH 5.0 as mobile phase B. Used and performed with the appropriate flow rate and gradient. The results of evaluation by cation exchange chromatography (IEC) are shown in FIG.
As shown in FIG. 41, not only humanized PM1 antibody (WT), which is an anti-IL-6 receptor antibody, but also F2H / L39, which is an anti-IL-6 receptor antibody, H0L0, which is an anti-IL-31 receptor antibody, and anti-RANKL. Also in the DNS, which is an antibody, it was confirmed that the heterogeneity increases by converting the constant region from IgG1 to IgG2, and that the heterogeneity can be reduced by converting the constant region to M14 or M58. It was. By changing the EU numbering cysteine at EU numbering 131 in the CH1 domain of the H chain and the 219th cysteine at EU numbering in the upper hinge of the H chain to serine, the antibody sequence and antigen type of the variable region can be changed. Nevertheless, it was shown that heterogeneity derived from native IgG2 can be reduced.

表１４に示したとおり、抗IL-31レセプター抗体であるH0L0、抗RANKL抗体であるDNSにおいても、定常領域をIgG1からM58に変換することで、抗IL-6レセプター抗体であるWT同様、ヒトFcRnへの結合活性が向上することが確認された。これより、可変領域の抗体配列および抗原の種類に関わらず、定常領域をIgG1からM58に変換することでヒトにおける血漿中滞留性が向上する可能性が示された。 [Example 17] Effect of improving the retention in plasma by a novel constant region M58 in various antibodies As shown in Example 15, in humanized PM1 antibody (WT), which is an anti-IL-6 receptor antibody, the constant region was changed from IgG1. By converting to M58, it was found that the binding activity to human FcRn was improved and the plasma retention was improved in human FcRn transgenic mice. Therefore, it was examined whether the retention in plasma can be improved by converting the constant region into M58 for IgG1 antibodies other than humanized PM1 antibodies.
As an antibody other than humanized PM1 antibody (WT), anti-IL-31 receptor antibody H0L0 (H0L0_VH amino acid sequence SEQ ID NO: 147, H0L0_VL amino acid sequence SEQ ID NO: 148), anti-RANKL antibody DNS (DNS_VH amino acid sequence sequence) No. 149, DNS_VL amino acid sequence SEQ ID NO: 150) was used. For each antibody, IgG1 (SEQ ID NO: 19) and M58 (SEQ ID NO: 151) were prepared as constant regions, and the binding activity to human FcRn was evaluated by the method shown in Example 14. The results are shown in Table 14.

As shown in Table 14, even in the anti-IL-31 receptor antibody H0L0 and the anti-RANKL antibody DNS, the constant region is converted from IgG1 to M58, as in the case of the anti-IL-6 receptor antibody WT. It was confirmed that the binding activity to FcRn was improved. From this, it was shown that the retention in plasma in humans can be improved by converting the constant region from IgG1 to M58 regardless of the antibody sequence of the variable region and the type of antigen.

WT-IgG1およびWT-IgG2のTm値はほぼ同等で約94℃程度（IgG2のほうが約1℃低い）であったのに対して、WT-SCおよびWT-CSのTm値は約86℃であり、WT-IgG1およびWT-IgG2と比較して著しくTm値が低下していた。一方、WT-M58、WT-SKSCのTm値は約94℃であり、ほぼWT-IgG1およびWT-IgG2と同等であった。WT-SCおよびWT-CSは安定性がIgG2と比較して著しく低いことから、医薬品として開発するためには、CH1ドメインのシステインもセリンに改変したWT-SKSCおよびWT-M58のほうが好ましいと考えられた。WT-SCおよびWT-CSのTm値がIgG2と比較して大幅に低下した理由として、WT-SCおよびWT-CSはIgG2のジスルフィド結合パターンとは異なる様式を取っているためと考えられた。
また、DSC変性曲線を比較した場合、WT-IgG1、WT-SKSC、WT-M58のFab部分の変性ピークはシャープかつ単一であったのに対して、WT-SCおよびWT-CSはこれらと比較して、Fab部分の変性ピークがブロードであり、WT-IgG2はFab部分の変性ピークの低温側にショルダーピークが認められた。DSCの変性ピークは単一成分の場合は通常シャープな変性ピークを示すが、Tmが異なる複数成分（つまりヘテロジェニティー）が存在する場合、変性ピークはブロードになると考えられる。すなわち、WT-IgG2、WT-SCおよびWT-CSには複数成分存在し、WT-SCおよびWT-CSは、天然型IgG2のヘテロジェニティーが十分低減されていない可能性が示唆された。このことから、野生型IgG2のヘテロジェニティーはヒンジ部分のシステインのみならず、CH1ドメインに存在するシステインの両方が関与していると考えられ、DSC上のヘテロジェニティーを低減するためにはヒンジ部分のシステインのみならず、CH1ドメインのシステインも改変する必要があると考えられた。また、上述のとおり、ヒンジ部分のシステインのみならず、CH1ドメインのシステインを改変することで初めて野生型IgG2と同等の安定性を有することが可能である。
以上より、IgG2のヒンジ領域に由来するヘテロジェニティーを低減した定常領域として、ヒンジ部分のシステインのみをセリンに置換した定常領域であるSCとCSはヘテロジェニティーおよび安定性の観点で不十分であると考えられ、ヒンジ部分のシステインに加えてCH1ドメインに存在するEUナンバリング131番目のシステインもセリンに置換することで初めてIgG2と同等の安定性を維持しつつヘテロジェニティーを大幅に低減することが可能であることが見出された。そのような定常領域としては、上述のM14、M31、M58、M73等が挙げられ、特にM58およびM73は血漿中滞留性が向上し、安定性が高く、ヘテロジェニティーが低減されていることから、抗体医薬品の定常領域として非常に有用であると考えられた。 [Example 18] Effect of cysteine of CH1 domain on heterogeneity and stability As shown in Example 8, with the aim of reducing the heterogeneity of native IgG2, the cysteine and CH1 of the hinge part of IgG2 Modification of cysteine present in the domain was performed. As a result of examination of various variants, among the wild type IgG2 constant region sequences, the EU numbering 131st cysteine and 133th arginine present in the CH1 domain of the H chain were changed to serine and lysine, respectively, and the H chain upper hinge It was found that SKSC (SEQ ID NO: 154), which is a constant region in which the 219th cysteine of the EU numbering 219, which is modified in serine, is modified to serine, can reduce heterogeneity without reducing stability. It was done.
On the other hand, as a method for reducing heterogeneity, a method in which only the EU numbering 219th cysteine present in the upper hinge of the H chain is changed to serine and a method in which only the 220th cysteine is changed to serine can be considered. SC (SEQ ID NO: 155), which is a constant region in which EU numbering 219th cysteine of IgG2 is modified to serine, and CS (SEQ ID NO: 156), which is a constant region in which EU2 numbering cysteine of IgG2 is modified to serine ) And WT-SC (SEQ ID NO: 157) and WT-CS (SEQ ID NO: 158) having a constant region, and heterogeneity with WT-IgG1, WT-IgG2, WT-SKSC and WT-M58, and Comparison of thermal stability was performed. Further, as an antibody other than WT, the constant region is IgG1 against F2H / L39 (F2H / L39_VH amino acid sequence SEQ ID NO: 145, F2H / L39_VL amino acid sequence SEQ ID NO: 146), which are different anti-IL-6 receptor antibodies. (SEQ ID NO: 19), IgG2 (SEQ ID NO: 20), SC (SEQ ID NO: 155), CS (SEQ ID NO: 156), SKSC (SEQ ID NO: 154), M2 (SEQ ID NO: 24) F2H / L39-IgG1, F2H / L39-IgG2, F2H / L39-SC, F2H / L39-CS, F2H / L39-SKSC, and F2H / L39-M14 were prepared and compared for heterogeneity and stability.
WT-IgG1, WT-IgG2, WT-SC, WT-CS, WT-SKSC, WT-M58 and F2H / L39-IgG1, F2H / L39-IgG2, F2H / L39-SC, F2H / L39-CS, F2H / As an evaluation method for heterogeneity of L39-SKSC and F2H / L39-M14, evaluation was performed by cation exchange chromatography. Using ProPac WCX-10 (Dionex) as the column, 20 mM Sodium Acetate, pH 5.0 as mobile phase A, 20 mM Sodium Acetate, 1M NaCl, pH 5.0 as mobile phase B, using the appropriate flow rate and gradient Carried out. The results of evaluation by cation exchange chromatography are shown in FIG.
As a result, as shown in FIG. 42, in both WT and F2H / L39, the heterogeneity was increased by converting the constant region from IgG1 to IgG2, but the constant region was converted to SKSC and M14 or M58. The heterogeneity was greatly reduced. On the other hand, when the constant region was SC, the heterogeneity was greatly reduced as in the case where the constant region was SKSC. However, when the constant region was CS, the heterogeneity was not sufficiently improved.
In general, in order to develop an antibody as a pharmaceutical product, it is desirable to have high stability in order to prepare a stable preparation in addition to low heterogeneity. Therefore, as an evaluation method of stability, the thermal denaturation intermediate temperature (Tm value) was evaluated by differential scanning calorimetry (DSC) (VP-DSC, manufactured by Microcal). Thermal denaturation intermediate temperature (Tm value) is an indicator of stability, and it is desirable that the thermal denaturation intermediate temperature (Tm value) is high in order to produce a stable pharmaceutical product (J Pharm Sci. 2008 Apr; 97 (4): 1414-26.). Dialyze (EasySEP, TOMY) WT-IgG1, WT-IgG2, WT-SC, WT-CS, WT-SKSC, WT-M58 against a solution of 20 mM sodium acetate, 150 mM NaCl, pH 6.0, about 0.1 DSC measurement was performed at a temperature increase rate of 1 ° C./min from 40 ° C. to 100 ° C. at a protein concentration of mg / mL. The resulting DSC denaturation curve is shown in FIG. 43, and the Tm value of the Fab portion is shown in Table 15 below.

The Tm values of WT-IgG1 and WT-IgG2 were about the same, about 94 ° C (IgG2 was about 1 ° C lower), whereas that of WT-SC and WT-CS was about 86 ° C. Yes, the Tm value was significantly reduced as compared with WT-IgG1 and WT-IgG2. On the other hand, the Tm value of WT-M58 and WT-SKSC was about 94 ° C., which was almost equivalent to WT-IgG1 and WT-IgG2. Since WT-SC and WT-CS are significantly less stable than IgG2, WT-SKSC and WT-M58, in which the cysteine of the CH1 domain is also changed to serine, are preferred for development as pharmaceuticals. It was. The reason why the Tm values of WT-SC and WT-CS were greatly reduced compared to IgG2 was considered to be because WT-SC and WT-CS took a different form from the disulfide bond pattern of IgG2.
In addition, when comparing DSC denaturation curves, the denaturation peaks in the Fab portion of WT-IgG1, WT-SKSC, and WT-M58 were sharp and single, whereas WT-SC and WT-CS In comparison, the denatured peak in the Fab portion was broad, and WT-IgG2 had a shoulder peak on the low temperature side of the denatured peak in the Fab portion. The DSC denaturation peak usually shows a sharp denaturation peak in the case of a single component, but it is considered that the denaturation peak becomes broad when there are multiple components having different Tm (that is, heterogeneity). That is, WT-IgG2, WT-SC and WT-CS have multiple components, suggesting that WT-SC and WT-CS may not sufficiently reduce the heterogeneity of natural IgG2. Therefore, it is considered that the heterogeneity of wild type IgG2 involves not only the cysteine in the hinge part but also the cysteine present in the CH1 domain. It was considered necessary to modify not only the partial cysteine but also the cysteine of the CH1 domain. Further, as described above, it is possible to have stability equivalent to that of wild-type IgG2 only by modifying not only the cysteine in the hinge portion but also the cysteine in the CH1 domain.
Based on the above, as constant regions with reduced heterogeneity derived from the IgG2 hinge region, SC and CS, which are constant regions in which only the cysteine in the hinge portion is replaced with serine, are insufficient in terms of heterogeneity and stability. It is believed that there is a significant reduction in heterogeneity while maintaining the same stability as IgG2 for the first time by substituting serine for the 131st EU numbering cysteine present in the CH1 domain in addition to the cysteine in the hinge part. Has been found to be possible. Examples of such a constant region include the aforementioned M14, M31, M58, M73 and the like. Particularly, M58 and M73 have improved plasma retention, high stability, and reduced heterogeneity. Therefore, it was considered to be very useful as a constant region of antibody drugs.

TOCILIZUMAB、コントロール、Fv3-M73、Fv4-M73、およびFv5-M83の等電点を当業者公知の方法により等電点電気泳動により測定した結果、TOCILIZUMABの等電点は約9.3、コントロールは約8.4〜8.5、Fv3-M73は約5.7〜5.8、Fv4-M73は約5.6〜5.7、Fv5-M83は5.4〜5.5であり、いずれの抗体もTOCILIZUMABおよびコントロールと比較して等電点が大幅に低下した。また、可変領域VH/VLの理論等電点をGENETYX(GENETYX CORPORATION)により計算したところ、TOCILIZUMABの理論等電点は9.20、コントロールは7.79、Fv3-M73は5.49、Fv4-M73は5.01、Fv5-M83は4.27であり、いずれの抗体もTOCILIZUMABおよびコントロールと比較して等電点が大幅に低下した。よって、Fv3-M73、Fv4-M73、およびFv5-M83はTOCILIZUMABおよびコントロールと比較して血漿中滞留性が向上していると考えられた。
TOCILIZUMAB、Fv3-M73、Fv4-M73、およびFv5-M83の可変領域配列に存在するT-cellエピトープをTEPITOPE（Methods. 2004 Dec;34(4):468-75）を用いて解析を行った。その結果、TOCILIZUMABは多くの配列がHLAに結合するT-cellエピトープが存在すると予測されたが、Fv3-M73、Fv4-M73、およびFv5-M83はT-cellエピトープに結合すると予測された配列が大幅に減少した。また、Fv3-M73、Fv4-M73、およびFv5-M83はフレームワークにマウス配列が残存せず完全ヒト化されている。これらのことから、Fv3-M73、Fv4-M73、およびFv5-M83の免疫原性はTOCILIZUMABと比較して大幅に免疫原性リスクが低減されている可能性が示唆された。 [Example 19] Production of fully humanized IL-6 receptor antibody with improved PK / PD
To produce a fully humanized IL-6 receptor antibody with improved PK / PD against TOCILIZUMAB (H chain WT-IgG1 (SEQ ID NO: 15), L chain WT (SEQ ID NO: 105)) Molecules were made.
In order to improve the ka of F2H-IgG1, the modification of affinity enhancement obtained in Example 2 was replaced with tryptophan number 35 by valine, and by number 50 tyrosine by phenylalanine, number 62 serine threonine Substitution was made. In addition, to reduce pI without increasing the risk of immunogenicity, the 102nd tyrosine was replaced with valine, the 105th glutamine was replaced with glutamic acid, the 107th isoleucine was replaced with threonine, and the constant region. VH5-M83 (SEQ ID NO: 139 (amino acid sequence)) was prepared by replacing IgG1 with M83. In addition, VL5-kappa (SEQ ID NO: 181 (amino acid sequence)) in which glutamine No. 27 was substituted with glutamine was prepared to improve L39 ka. Furthermore, as a result of carrying out various screenings by producing TOCILIZUMAB variants in which multiple mutations in the variable region and constant region of TOCILIZUMAB found in the above Examples and newly found mutations were combined, fully humanized IL was obtained. -6 receptor antibodies include Fv3-M73 (H chain VH4-M73 SEQ ID NO: 182; L chain VL1-kappa SEQ ID NO: 183), Fv4-M73 (H chain VH3-M73 SEQ ID NO: 180, L chain VL3-kappa SEQ ID NO: 181), Fv5-M83 (H chain VH5-M83 SEQ ID NO: 139, L chain VL5-kappa SEQ ID NO: 138) were found.
The affinity of the prepared Fv3-M73, Fv4-M73, and Fv5-M83 for IL-6 receptor was compared with that of TOCILIZUMAB (refer to the reference example for the method). Table 16 shows the results of measuring the affinity of these antibodies for IL-6 receptor. In addition, the neutralizing activity of BaF / gp130 was compared with TOCILIZUMAB and control (known high affinity high IL-6 receptor antibody of reference example, VQ8F11-21 hIgG1 in US 2007/0280945) (refer to reference example for method). The results of measuring the biological activity of these antibodies by BaF / gp130 are shown in FIG. 44 (IL-6 final concentration 300 ng / mL: TOCILIZUMAB, control, Fv5-M83) and FIG. 45 (IL-6 final concentration 30 ng / mL: TOCILIZUMAB, Fv3-M73, Fv4-M73). As shown in Table 16, Fv3-M73 and Fv4-M73 have an affinity about 2-3 times stronger than TOCILIZUMAB, and Fv5-M83 showed an affinity about 100 times stronger than TOCILIZUMAB (Fv5 Since it was difficult to measure affinity with -M83, the affinity was measured using Fv5-IgG1 in which the constant region was IgG1, and it is considered that the constant region generally does not affect the affinity). In addition, as shown in FIG. 45, Fv3-M73 and Fv4-M73 show a slightly stronger activity compared to TOCILIZUMAB, and as shown in FIG. 44, Fv5-M83 has a 50% inhibitory concentration of 100 times or more as compared with TOCILIZUMAB. It had a strong activity and showed a neutralization activity about 10 times higher as a 50% inhibitory concentration than the control which is a known high affinity high IL-6 receptor antibody.

The isoelectric point of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 was measured by isoelectric focusing by a method known to those skilled in the art, and as a result, the isoelectric point of TOCILIZUMAB was about 9.3, and the control was about 8.4. -8.5, Fv3-M73 is about 5.7-5.8, Fv4-M73 is about 5.6-5.7, Fv5-M83 is 5.4-5.5, and the isoelectric point of each antibody was greatly reduced compared to TOCILIZUMAB and control . In addition, when the theoretical isoelectric point of variable region VH / VL was calculated by GENETYX (GENETYX CORPORATION), the theoretical isoelectric point of TOCILIZUMAB was 9.20, control was 7.79, Fv3-M73 was 5.49, Fv4-M73 was 5.01, Fv5- M83 was 4.27, and the isoelectric point of all antibodies was greatly reduced compared to TOCILIZUMAB and control. Therefore, Fv3-M73, Fv4-M73, and Fv5-M83 were considered to have improved plasma retention compared to TOCILIZUMAB and controls.
T-cell epitopes present in the variable region sequences of TOCILIZUMAB, Fv3-M73, Fv4-M73, and Fv5-M83 were analyzed using TEPITOPE (Methods. 2004 Dec; 34 (4): 468-75). As a result, TOCILIZUMAB was predicted to have a T-cell epitope in which many sequences bind to HLA, while Fv3-M73, Fv4-M73, and Fv5-M83 had sequences predicted to bind to a T-cell epitope. It decreased significantly. Fv3-M73, Fv4-M73, and Fv5-M83 are completely humanized with no mouse sequence remaining in the framework. These results suggested that the immunogenicity risk of Fv3-M73, Fv4-M73, and Fv5-M83 may be greatly reduced compared to TOCILIZUMAB.

[Example 20] Monkey PK / PD test of fully humanized IL-6 receptor antibody
TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 were administered to cynomolgus monkeys at a dose of 1 mg / kg intravenously and the plasma concentration was evaluated (see Reference Examples for methods). FIG. 46 shows changes in plasma concentration after intravenous administration of TOCILIZUMAB, Fv3-M73, Fv4-M73, and Fv5-M83. As a result, all of Fv3-M73, Fv4-M73, and Fv5-M83 significantly improved plasma retention in cynomolgus monkeys compared to TOCILIZUMAB and controls. In particular, the plasma retention of Fv3-M73 and Fv4-M73 was significantly improved compared to TOCILIZUMAB.
Cynomolgus monkey IL-6 5 μg from day 6 to day 18 of antibody administration (from day 3 to day 10 for TOCILIZUMAB) to evaluate the efficacy of neutralization of cynomolgus membrane IL-6 receptor / kg was subcutaneously administered daily to the back of the lumbar region, and the CRP concentration of each individual was measured 24 hours later (see Reference Examples for methods). The change in CRP concentration at the time of administration of each antibody is shown in FIG. In order to evaluate the degree of neutralization of cynomolgus monkey soluble IL-6 receptor, the concentration of unbound cynomolgus monkey soluble IL-6 receptor in cynomolgus monkey plasma was measured and soluble IL- The neutralization rate of 6 receptors was calculated (refer to Reference Example for the method). The transition of the neutralization rate of soluble IL-6 receptor upon administration of each antibody is shown in FIG.
Fv3-M73, Fv4-M73, and Fv5-M83 all neutralize cynomolgus monkey membrane IL-6 receptor more persistently than TOCILIZUMAB and the known high affinity anti-IL-6 receptor antibody control. The increase of CRP was suppressed for a long time. In addition, Fv3-M73, Fv4-M73, and Fv5-M83 all neutralize cynomolgus monkey soluble IL-6 receptor more persistently than TOCILIZUMAB and control, and non-binding cynomolgus monkey soluble IL- 6 receptors increased for a long time. As a result, Fv3-M73, Fv4-M73, and Fv5-M83 are all superior to TOCILIZUMAB and controls in terms of the persistence of neutralization of membrane-type IL-6 receptor and soluble IL-6 receptor. It was found. In particular, the sustainability of neutralization of Fv3-M73 and Fv4-M73 was extremely excellent. On the other hand, Fv5-M83 suppresses CRP and non-binding cynomolgus monkey soluble IL-6 receptor to a lower level than Fv3-M73 and Fv4-M73. Type 6 IL-6 receptor was thought to be more strongly neutralized than Fv3-M73 and Fv4-M73 and the known high affinity anti-IL-6 receptor antibody control. This is probably because Fv5-M83 has a stronger affinity for the IL-6 receptor than the control, and the biological activity of BaF / gp130 is reflected in cynomolgus monkeys in vivo.
Therefore, compared to TOCILIZUMAB and controls, Fv3-M73 and Fv4-M73 have extremely long-lasting effects as anti-IL-6 receptor neutralizing antibodies, and the administration frequency and dosage are greatly reduced. In addition, it was found that Fv5-M83 is extremely excellent in the strength of action as an anti-IL-6 receptor neutralizing antibody, and also excellent in durability of action. Therefore, Fv3-M73, Fv4-M73, and Fv5-M83 are considered useful as pharmaceutical agents as IL-6 antagonists.

[Reference example]
Preparation of recombinant cynomolgus monkey soluble IL-6 receptor (cIL-6R) Published rhesus monkey IL-6 receptor gene sequence (Birney et al, Ensembl 2006, Nucleic Acids Res. 2006 Jan 1; 34 (Database issue): D556 Based on -61.), An oligo DNA primer was prepared, and a DNA fragment encoding the full length of the cynomolgus IL-6 receptor gene was prepared by PCR using the cDNA prepared from cynomolgus monkey pancreas as a template. The obtained DNA fragment was inserted into an animal cell expression vector, and a CHO constant expression strain (cyno.sIL-6R-producing CHO cell) was prepared using this. After purifying the culture fluid of cyno.sIL-6R-producing CHO cells using HisTrap column (GE Healthcare Bioscience), it is concentrated using Amicon Ultra-15 Ultracel-10k (Millipore), and Superdex200pg16 / 60 gel filtration column (GE Healthcare). The product was further purified by Care Bioscience) to obtain a final purified product of soluble cynomolgus monkey IL-6 receptor (hereinafter cIL-6R).

Preparation of recombinant cynomolgus monkey IL-6 (cIL-6) Cynomolgus monkey IL-6 was prepared as follows. A base sequence encoding 212 amino acids registered in SWISSPROT Accession No.P79341 was created, cloned into an animal cell expression vector, and introduced into CHO cells to produce a constant expression cell line (cyno.IL-6 production) CHO cells). After purifying the culture fluid of cyno.IL-6 producing CHO cells using SP-Sepharose / FF column (GE Healthcare Bioscience), concentrating using Amicon Ultra-15 Ultracel-5k (Millipore), Superdex75pg26 / 60 gel filtration Further purification was performed using a column (GE Healthcare Bioscience), and concentration was performed using Amicon Ultra-15 Ultracel-5k (Millipore) to obtain a final purified product of cynomolgus IL-6 (hereinafter, cIL-6).

Preparation of known high-affinity anti-IL-6 receptor antibody As a known high-affinity anti-IL-6 receptor antibody, VQ8F11-21 hIgG1 which is a high-affinity anti-IL-6 receptor antibody described in US 2007/0280945 A1 In order to express (US 2007/0280945 A1, H chain amino acid sequence: SEQ ID NO: 19, L chain amino acid sequence: SEQ ID NO: 27), an animal cell expression vector was constructed. The antibody variable region was prepared by a PCR method combining synthetic oligo DNA (assembly PCR), and IgG1 was used for the constant region. The antibody variable region and the constant region were combined by Assembly PCR method and inserted into an animal expression vector to prepare the intended H chain expression vector and L chain expression vector. The base sequence of the obtained expression vector was determined by a method known to those skilled in the art. Expression and purification were performed using the prepared expression vector. Expression and purification were performed by the method described in Example 1 to obtain a high affinity anti-IL-6 receptor antibody (hereinafter referred to as control).

Evaluation of binding to IL-6 receptor by Biacore
Kinetic analysis of antigen-antibody reaction was performed using Biacore T100 (GE Healthcare). An appropriate amount of anti-IgG (γ-chain specific) F (ab ′) ₂ is immobilized on the sensor chip by the amine coupling method, then the target antibody is bound at pH 7.4, and various antibodies are further immobilized at pH 7.4. SR344, an IL-6 receptor adjusted to a concentration, was run as an analyte, and the interaction between the antibody and SR344 was measured. All measurements were performed at 37 ° C. From the sensorgram obtained from the measurement, the association rate constant k _a (1 / Ms) and dissociation rate constant k _d (1 / s), which are kinetic parameters, are calculated, and K _D (M ) Was calculated. Biacore T100 Evaluation Software (GE Healthcare) was used for calculation of each parameter.

Measurement of Antibody Plasma Concentration, CRP Concentration, and Unbound Soluble IL-6 Receptor by Monkey PK / PD Test Cynomolgus monkey plasma concentration was measured by ELISA using methods known to those skilled in the art.
The CRP concentration was measured with Cias R CRP (Kanto Chemical Co., Inc.) using an automatic analyzer (TBA-120FR, Toshiba Medical Systems Co., Ltd.).
The concentration of unbound cynomolgus monkey soluble IL-6 receptor in cynomolgus monkey plasma was measured as follows. By adding 30 μL of cynomolgus monkey plasma to an appropriate amount of rProtein A Sepharose Fast Flow (GE Healthcare) resin dried in a 0.22 μm filter cup (Millipore), all IgG-type antibodies (cynomolgus IgG, anti-human) IL-6 receptor antibody and anti-human IL-6 receptor antibody-cynomolgus monkey soluble IL-6 receptor complex) were adsorbed to Protein A. Then, it spin-down with the high-speed centrifuge and collect | recovered pass solutions. Since the path solution does not contain anti-human IL-6 receptor antibody-cynomolgus monkey soluble IL-6 receptor complex bound to protein A, the concentration of cynomolgus monkey soluble IL-6 receptor in the protein A path solution should be measured. Can measure the concentration of unbound soluble IL-6 receptor. Cynomolgus monkey soluble IL-6 receptor concentration is measured by a method known to those skilled in the art to measure human IL-6 receptor concentration using the cynomolgus monkey soluble IL-6 receptor (cIL-6R) prepared above as a standard. did. The neutralization rate of soluble IL-6 receptor was calculated by the following formula.
(Unbound soluble IL-6 receptor concentration after antibody administration / soluble IL-6 receptor concentration before antibody administration) × 100
[Example 21]
(1) Production of point mutation gene of humanized H0L0 antibody
Various point mutation genes were prepared using the gene encoding the glypican 3 antibody containing CDR of the humanized GC33 antibody disclosed in WO2006 / 046751 as a starting material. Oligo DNAs designed based on the normal and reverse strand sequences including the modification site were synthesized. A plurality of point mutation genes were prepared using a commercially available QuikChange Site-Directed Mutagenesis Kit (Stratagene). The point mutation gene was prepared by PCR according to the following conditions. After heating a reaction mixture consisting of 10 ng template plasmid, 10 pmol of normal and reverse synthetic oligo DNA, 10x Buffer, dNTP mix and Pfu turbo DNA polymerase attached to the kit at 95 ° C for 30 seconds A PCR reaction cycle consisting of 30 seconds at 95 ° C., 1 minute at 55 ° C. and 4 minutes at 68 ° C. was performed 18 times. After DpnI attached to the kit was added to the reaction mixture, a restriction digestion reaction with a restriction enzyme was continued at 37 ° C. for 1 hour. As a result of transformation of DH5α competent cells (TOYOBO) with the reaction solution, a transformant was obtained. Based on the determination of the nucleotide sequence of the plasmid DNA isolated from the transformant, the point mutation gene confirmed to have been introduced with the point mutation is contained in an expression vector that enables expression of the inserted gene in animal cells. Cloned. The modified gene was obtained by modification having the following constitution.

Transient expression of the humanized H0L0 antibody and its point mutation modified antibody was performed by transient expression using Polyethyleneimine (Polysciences Inc.). HEK293 cells exfoliated with Trypsin EDTA (Invitrogen) were seeded in a 10 cm ² culture dish at 6 × 10 ⁶ cells / 10 mL. The next day, 4.6 μg H chain expression plasmid DNA and 9.2 μg L chain expression plasmid DNA were mixed with 690 μl SFMII medium and 20.8 μg Polyetyleneimine, and the mixture was allowed to stand at room temperature for 10 minutes. It was. The total amount of the mixed solution was dropped onto a culture dish in which HEK293 cells were seeded as described above. The purified humanized H0L0 antibody and its point mutation-modified antibody were purified from the culture supernatant collected about 72 hours later using rProteinA sepharose ™ Fast Flow (GE Healthcare) according to the procedure manual.

(1-1) Modified heat denaturation intermediate temperature (Tm) of Tm value of humanized H0L0 antibody is a denaturation in a thermogram (Cp vs. T) obtained after heating a test sample solution at a constant programmed heating rate. It is grasped as the peak apex. The Tm value of the humanized H0L0 antibody was measured by preparing a sample solution for DSC measurement as follows. First, a 20 mol / l sodium acetate buffer solution (pH 6.0) containing 150 mmol / l sodium chloride is applied to the dialysis membrane, and an antibody solution equivalent to 50-100 μg enclosed in a dialysis membrane During dialysis. Thereafter, a sample solution prepared using an external solution for dialysis so that the antibody concentration was 50-100 μg / ml was used as a sample solution for DSC measurement.

  A suitable DSC apparatus, such as DSC-II (Calorimetry Sciences Corporation), is preferably used for this experiment. After the sample solution and the reference solution (external dialysis solution) prepared by the above method were sufficiently degassed, each test sample was enclosed in a calorimeter cell and heat-equilibrium was performed at 40 ° C. A DSC scan was then performed from 40 ° C to 100 ° C at a scan rate of about 1 K / min. The result of the measurement is expressed as the apex of the denaturing peak as a function of temperature. Fab domain peak assignment was performed with reference to non-patent literature (Rodolfo et al., Immunology Letters (1999), 47-52), and the thermal denaturation intermediate temperature of the humanized H0L0 antibody was calculated. As a specific example, a chart obtained from DSC (Differential Scanning Calorimetry) measurement of Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody is illustrated in FIG.

  The Tm value of the humanized H0L0 antibody consisting of the H chain represented by SEQ ID NO: 195 and the L chain represented by SEQ ID NO: 201 based on the calculation by the method described above is 76.6 ° C. The Tm values for the exemplified Synagis and Herceptin are measured as 85.4 ° C and 81.8 ° C, respectively. Therefore, it was shown that the Tm value of the humanized H0L0 antibody is lower than that of the existing antibody.

  Therefore, a modified antibody of humanized H0L0 antibody was prepared for the purpose of improving its Tm value. H15 (SEQ ID NO: 196) in which the V37I, A40P, M48I, and L51I modifications that modify the VH1b subclass to the VH4 subclass are added to the H2 FR2 of the H0L0 antibody represented by SEQ ID NO: 195 is prepared. It was done. Its Tm value was improved to 79.1 ° C. Modification of L42Q, S48A, Q50R, which changes FR2 of the L chain of the H0L0 antibody represented by SEQ ID NO: 201 from VK2 to VK3, and V2I, which replaces V2 of FR1 with I, which is a germline sequence The modified L4 (SEQ ID NO: 202) was produced. Its Tm value was improved to 77.2 ° C. The Tm value of the H15L4 antibody in which these two variants were combined was improved to 80.5 ° C.

(1-2) Modification of pI value of humanized H0L0 antibody When the pI value of an antibody decreases, the blood half-life of the antibody is extended. In contrast, an increase in the pI value of the antibody improves the tissue transferability of the antibody. It is not known whether either an increase or decrease in the pI value of an antibody that exerts an effect on cancer treatment results in an enhanced tumor suppressive effect. Therefore, by producing a modified antibody of the humanized H0L0 antibody with a decreased pI value and a modified antibody of the humanized H0L0 antibody with an increased pI value, and comparing the antitumor effects of the two, tumor modification with either modification is high. It was verified whether it would be effective.

The pI value of each antibody was calculated based on analysis by isoelectric focusing. The electrophoresis was performed as follows. Phast-Gel Dry IEF (AmerchamBioscience) gel was swollen for about 60 minutes with a swelling liquid having the following composition using Phastsystem Cassette (AmerchamBioscience).
(A) Composition of swelling liquid for high pI:
1.5 ml 10% Glycerol
100 μl Pharmalyte 8-10.5 for IEF (AmerchamBioscience)
(B) Composition of swelling liquid for low pI:
1.5 ml purified water
20 μl Pharmalyte 8-10.5 for IEF (AmerchamBioscience)
80 μl Pharmalyte 5-8 for IEF (AmerchamBioscience)

About 0.5 μg of antibody was applied to the swollen gel and isoelectric focusing was performed by using PhastSystem (AmerchamBioscience) controlled by the following program. The sample was added to the gel at Step 2 in the program below. The Calibration Kit for pI (AmerchamBioscience) was used as the pI marker.
Step 1: 2000 V, 2.5 mA, 3.5 W, 15 ° C, 75 Vh
Step 2: 200 V, 2.5 mA, 3.5 W, 15 ° C, 15 Vh
Step 3: 2000 V, 2.5 mA, 3.5 W, 15 ° C, 410 Vh

  After electrophoresis, the gel was immobilized with 20% TCA, and then silver staining was performed using Silver staining Kit, protein (AmerchamBioscience) according to the procedure attached to the kit. After staining, the isoelectric point of each antibody as a test sample was calculated based on the known isoelectric point of the pI marker. FIGS. 50 and 51 show a migration image of high pI isoelectric focusing and a migration image of low pI isoelectric focusing, respectively.

(A) Modification with increased pI value
Hspu2.2 (Hu2.2) (SEQ ID NO: 200) in which Q43K, D52N, and Q107R were further modified in H15 was produced. In addition, Lspu2.2 (Lu2.2) (SEQ ID NO: 206) was prepared by modifying S25A by replacing E25Q, Q27R, Q105R, and S25 of CDR2 with a large A in the germline in L4. The Tm value of the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody, which is an antibody consisting of Hspu2.2 (Hu2.2) and Lspu2.2 (Lu2.2), is measured as 76.8 ° C, and the pI value is 9.6. It was measured. Since the pI value of the H0L0 antibody was 8.9, the pI value of the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody increased by 0.7.

(B) Modification with reduced pI value
Hspd1.8 (Hd1.8) (SEQ ID NO: 199) was prepared by further modifying K15T, Q43E, K63S, K65Q, and G66D on H15. Lspd1 with L4 modified Q27E, KISRVE, FR3 79-84 sequence comprising L4, modified to TISSLQ, and modified S25A as well as modified Lspu2.2 (Lu2.2). 6 (Ld1.6) (SEQ ID NO: 205) was produced. The Tm value of the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody, which is an antibody consisting of Hspd1.8 (Hd1.8) and Lspd1.6 (Ld1.6), was measured as 72.6 ° C., and the pI value was 7.4. It was measured. Since the pI value of the H0L0 antibody was 8.9, the pI value of the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody was decreased by 1.5.

(2) Evaluation of binding activity of point mutation modified antibody of H0L0 antibody by competitive ELISA Evaluation of the H0L0 antibody purified in (1) and its point mutation modified antibody by competitive ELISA was performed. A soluble GPC3 core polypeptide (SEQ ID NO: 207) prepared to 1 μg / ml was added to a 96-well plate at 100 μl per well. The plate was allowed to stand overnight at 4 ° C., and the soluble GPC3 core polypeptide was immobilized on the plate. The soluble GPC3 core polypeptide immobilized on the plate was washed 3 times with wash buffer using Skan WASHER400 (Molecular Devices), 200 μl blocking buffer was added, and blocked at 4 ° C overnight or longer. It was done. The plate on which the soluble GPC3 core polypeptide had been immobilized and blocked was then washed three times with wash buffer using SkanWASHER400. Thereafter, 100 μl of a mixture of various concentrations of H0L0 antibody or its point mutation-modified antibody and biotinylated H0L0 antibody at a final concentration of 0.3 μg / ml was added per well of the plate. Biotinylation of the H0L0 antibody was performed using the Biotin Labeling kit (Roche) according to the kit procedure. The plate was allowed to stand at room temperature for 1 hour, and then washed 5 times with a washing buffer using Skan WASHER400 (Molecular Devices). The plate containing 100 μl of Goat anti streptabidin Alkaline phosphatase (ZYMED) diluted 20,000 times with the substrate buffer per well was allowed to stand at room temperature for 1 hour, and then washed with Skan WASHER400. Washed 5 times. Phosphatase Substrate (Sigma) was prepared to 1 mg / ml using a substrate buffer, and 100 μl per well was added and allowed to stand for 1 hour. Using Benchmark Plus (BIO-RAD), the absorbance at 405 nm of the reaction solution in each well was measured using a control absorbance of 655 nm.

  As shown in FIG. 52, it was shown that the binding activity of the H15L4 antibody to the antigen was almost equivalent to that of the H0L0 antibody subjected to the modification. Further, as shown in FIG. 53, it was shown that the binding activity of the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody to the antigen was almost equivalent to that of the H0L0 antibody subjected to the modification. Furthermore, as shown in FIG. 54, it was shown that the binding activity of the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody to the antigen was almost equivalent to that of the H0L0 antibody subjected to the modification.

[Reference Example 22] Disruption of fucose transporter gene in CHO cells
(1) Construction of targeting vector
(1-1) Preparation of KO1 vector
By PCR with pc of Hyg5-BH and Hyg3-NT from pcDNA3.1 / Hygro (Invitrogen), by adding the BamHI site and TGCGC sequence 5 'to the start codon of the Hygromycin resistance gene (Hygr) Hygr was extracted by adding the Not I site to the 3 ′ side including the region up to the SV40 polyA addition signal, with the same sequence as the 5 ′ side of the start codon of the fucose transporter gene.
Forward primer
Hyg5-BH 5'-GGATCCTGCGCATGAAAAAGCCTGAACTCACC -3 '(SEQ ID NO: 208)
Reverse primer
Hyg3-NT 5'-GCGGCCGCCTATTCCTTTGCCCTCGGACG-3 '(SEQ ID NO: 209)

  The fucose transporter targeting vector ver.1 (hereinafter referred to as KO1 vector) is the pMC1DT-A vector (Yagi T, Proc. Natl. Acad. Sci. USA (1990) 87, 9918-22,) and the fucose transporter. 5 'side (No.2,780 SmaI to No.4,232 BamHI of the base sequence shown in SEQ ID NO: 210), 3' side (No.4,284 to No.10,934 Sac I), and Hygr fragment are inserted respectively. It was built by doing. Since the promoter is not added to Hygr, the vector is characterized in that Hygr is expressed by the fucose transporter promoter when homologous recombination occurs. However, even if only one copy of a vector is introduced into a cell by homologous recombination, Hygr does not always express enough to acquire resistance to hygromycin B. The KO1 vector was cleaved with Not I and introduced into cells. The KO1 vector causes the fucose transporter to lose 41 base pairs of exon 1 including the start codon and lose function.

(1-2) Preparation of pBSK-pgk-1-Hygr
The mouse pgk-1 gene promoter was excised from the pKJ2 vector (Popo H, Biochemical Genetics (1990) 28, 299-308,) with EcoR I-Pst I and cloned into the EcoR I-Pst I site of pBluescript (Stratagene). PBSK-pgk-1 was prepared. Hygr added the EcoT22 I site and Kozak sequence to the 5 'side of Hygr by PCR using the primers of Hyg5-AV and Hyg3-BH from pcDNA3.1 / Hygro, 3' including the region up to the SV40 polyA addition signal. Hygr was extracted by adding a BamHI site on the side.
Forward primer
Hyg5-AV 5'- ATGCATGCCACCATGAAAAAGCCTGAACTCACC -3 '(SEQ ID NO: 211)
Reverse primer
Hyg3-BH 5'- GGATCCCAGGCTTTACACTTTATGCTTC -3 '(SEQ ID NO: 212)

  This Hygr (EcoT22 I-BamHI) fragment was inserted into the Pst I-BamHI site of pBSK-pgk-1 to prepare pBSK-pgk-1-Hygr.

(1-3) Preparation of KO2 vector The fucose transporter targeting vector ver.2 (hereinafter referred to as KO2 vector) is the pMC1DT-A vector on the 5 ′ side of the fucose transporter (No. of the nucleotide sequence shown in SEQ ID NO: 210). 2,780 Sma I to No. 4,232 BamH I), 3 ′ side (from No. 4,284 to No. 10,934 SacI), and pgk-1-Hygr fragment were each inserted. Unlike the KO1 vector, since the pgk-1 gene promoter is added to Hygr, the KO2 vector acquires resistance to hygromycin B even if only one copy of the vector is introduced into the cell by homologous recombination. The KO2 vector was cleaved with Not I and introduced into cells. The KO2 vector causes the fucose transporter to lose 46 base pairs of exon 1 including the start codon and lose function.

(1-4) Preparation of pBSK-pgk-1-Puror
The pPUR vector (BD Biosciences) was cleaved with Pst I and BamH I, and the excised fragment (Puror) was inserted into the Pst I-BamH I site of pBSK-pgk-1 to prepare pBSK-pgk-1-Puror. .

(1-5) Preparation of KO3 Vector The fucose transporter targeting vector ver.3 (hereinafter referred to as KO3 vector) is pMC1DT-A vector and the 5 ′ side of the fucose transporter (No. of the nucleotide sequence shown in SEQ ID NO: 210). 2,780 Sma I to No. 4,232 BamH I), 3 ′ side (from No. 4,284 to No. 10,934 SacI), and pgk-1-Puror fragment were respectively inserted. A sequence to which a screening primer shown below was bound was added in advance to the 3 ′ end of pgk-1-Puror. The KO3 vector was cleaved with Not I and introduced into cells. The KO3 vector causes the fucose transporter to lose 46 base pairs of exon 1 including the start codon and lose function.
Reverse primer
RSGR-A 5'-GCTGTCTGGAGTACTGTGCATCTGC-3 '(SEQ ID NO: 213)
Using the above three types of targeting vectors, we tried to knockout the fucose transporter gene.

(2) Introduction of vector into CHO cells
HT Supplement (100x) (Invitrogen) and penicillin streptomycin (Invitrogen) were added to CHO-S-SFMII HT- (Invitrogen) in an amount of 1/100 of the amount of CHO-S-SFMII HT-, respectively. This was subcultured with the DXB11 strain of CHO cells as a culture medium (hereinafter referred to as SFMII (+)), and further cultured after this gene transfer with this SFMII (+). 8 × 10 ⁶ CHO cells were suspended in 0.8 ml Dulbecco's phosphate buffer (hereinafter abbreviated as PBS, Invitrogen). 30 μg of the targeting vector was added to the cell suspension, and the cell suspension was transferred to Gene PulserCuvette (4 mm) (BioRad). After leaving on ice for 10 minutes, the vector was introduced into the cells by the electroporation method with GENE-PULSER II (BioRad) under the conditions of 1.5 kV and 25 μFD. After introducing the vector, the cells were suspended in 200 ml of SFMII (+) medium, and the cells were seeded at 100 μl / well in 20 96-well flat bottom plates (Iwaki). The plate was incubated for 24 hours at 37 ° C. in a CO ₂ incubator before adding the drug.

(3) First stage of knockout
KO1 vector or KO2 vector was introduced into each CHO cell, and selection with hygromycin B (Invitrogen) was performed 24 hours after the introduction of the vector. Hygromycin B was dissolved in SFMII (+) to 0.3 mg / ml and added at 100 μl / well.

(4) Screening of homologous recombinants by PCR
(4-1) Preparation of PCR Samples Homologous recombinants were screened by the PCR method. CHO cells to be used for screening should be cultured in 96-well flat-bottomed plates. After removing the culture supernatant, add 50 μl / well of cell lysis buffer, heat at 55 ° C for 2 hours, and then heat at 95 ° C for 15 minutes. Thus, proteinase K was inactivated and used as a PCR template. The cell lysis buffer was 10 μL LA well II (supplied with Takara LATAq) 5 μl, 2.5% 10% NP-40 (Roche), 4 μl proteinase K (20 mg / ml, Takara), and distilled water. (Nacalai Tesque) is composed of 38.5μl.

(4-2) PCR conditions
PCR reaction mixture is 1 μl of the above PCR sample, 5 μl of 10 × LA buffer II, 5 μl of MgCl 2 (25 mM), 5 μl of dNTP (2.5 mM), 2 μl of primer (each 10 μM), 0.5 μl of LA Taq (5 IU / μl), And 29.5 μl of distilled water (total of 50 μl). TP-F4 and THygro-R1 were used as PCR primers for screening cells transfected with KO1 vector, and TP-F4 and THygro-F1 were used as PCR primers for screening cells transfected with KO2 vector.

  PCR of cells transfected with KO1 vector was pre-heated at 95 ° C for 1 minute, 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 60 ° C for 2 minutes for 40 cycles, and 72 ° C 7 minutes double heating. Screening for cells transfected with KO2 vector: preheating at 95 ° C for 1 minute, 40 cycles of amplification cycle of 95 ° C for 30 seconds and 70 ° C for 3 minutes, and double heating at 70 ° C for 7 minutes It was.

The primers are as follows. In a sample of cells that have undergone homologous recombination, about 1.6 kb of DNA is amplified for the KO1 vector and about 2.0 kb of DNA is amplified for the KO2 vector. As for the primer, TP-F4 was set outside the vector and in the 5'-side fucose transporter genomic region, and THygro-F1 and THygro-R1 were set in Hygr in the vector.
Forward primer (KO1, KO2)
TP-F4 5'-GGAATGCAGCTTCCTCAAGGGACTCGC-3 '(SEQ ID NO: 214)
Reverse primer (KO1)
THygro-R1 5'-TGCATCAGGTCGGAGACGCTGTCGAAC -3 '(SEQ ID NO: 215)
Reverse primer (KO2)
THygro-F1 5'-GCACTCGTCCGAGGGCAAAGGAATAGC -3 '(SEQ ID NO: 216)

(5) PCR screening results
918 cells into which the KO1 vector was introduced were analyzed, of which one cell was considered to be a homologous recombinant (the homologous recombination efficiency was about 0.1%). In addition, 537 cells into which the KO2 vector was introduced were analyzed, of which 17 cells were considered to be homologous recombinants (homologous recombination efficiency was about 3.2%).

(6) Southern blot analysis Further confirmation was performed by the Southern blot method. From the cultured cells, 10 μg of genomic DNA was prepared according to a conventional method, and Southern blotting was performed. From the region of No.2,113-No.2,500 of the base sequence shown in SEQ ID NO: 210, a 387 bp probe was prepared by PCR using the following two kinds of primers, and this was used for confirmation by Southern blotting. Genomic DNA was cut with Bgl II.
Forward primer
Bgl-F: 5'-TGTGCTGGGAATTGAACCCAGGAC-3 '(SEQ ID NO: 217)
Reverse primer
Bgl-R: 5′-CTACTTGTCTGTGCTTTCTTCC-3 ′ (SEQ ID NO: 218)

  Cleavage with Bgl II resulted in a band of approximately 3.0 kb from the fucose transporter chromosome, approximately 4.6 kb from the chromosome that had undergone homologous recombination with the KO1 vector, and approximately 5.0 kb from the chromosome that had undergone homologous recombination with the KO2 vector. Appear. One to seven types of cells that had undergone homologous recombination with the KO1 vector and KO2 vector were used in the experiment. The only cell obtained with the KO1 vector was named 5C1, but later analysis revealed that it was composed of multiple cell populations, so it was cloned by limiting dilution and used in subsequent experiments. One of the cells obtained with the KO2 vector was named 6E2.

(7) Second stage of knockout
We tried to establish a cell line completely lacking the fucose transporter gene using three types of vectors for cells successfully homologously recombined with the KO1 and KO2 vectors. Combinations of vectors and cells are as follows. Method 1: KO2 vector and 5C1 cells (KO1), Method 2: KO2 vector and 6E2 cells (KO2), Method 3: KO3 vector and 6E2 cells (KO2). The vector was introduced into each cell, and selection with hygromycin B and puromycin (Nacalai Tesque) was started 24 hours after the introduction of the vector. Hygromycin B was adjusted to a final concentration of 1 mg / ml in Method 1 and 7 mg / ml in Method 2. Further, in method 3, the final concentration of hygromycin B was 0.15 mg / ml, and the final concentration of puromycin was 8 μg / ml.

(8) Preparation of screening samples for homologous recombinants by PCR is as described above. In the screening for method 1, both PCRs were performed to detect cells that had undergone homologous recombination with the KO1 vector and KO2 vector. For method 2, the following PCR primers were designed. TPS-F1 was set in the region of No. 3,924-3,950 of the nucleotide sequence shown in SEQ ID NO: 210, and SHygro-R1 was set in No. 4,248-4,274. This PCR primer amplifies 350 bp of the gene region of the fucose transporter that is deleted by the KO2 vector. Therefore, in the PCR screening in Method 2, cells in which 350 bp were not amplified were considered as cells completely lacking the fucose transporter gene. The PCR conditions were preheating at 95 ° C for 1 minute, 35 cycles of amplification cycle of 95 ° C for 30 seconds, 70 ° C for 1 minute, and double heating at 70 ° C for 7 minutes.
Forward primer
TPS-F1: 5′-CTCGACTCGTCCCTATTAGGCAACAGC-3 ′ (SEQ ID NO: 219)
Reverse primer
SHygro-R1: 5'-TCAGGGGCAGTGGAGCCTCCAGTCAGC-3 '(SEQ ID NO: 220)

  For method 3, TP-F4 was used as the forward primer and RSGR-A was used as the reverse primer. PCR conditions include preheating at 95 ° C for 1 minute, 35 cycles of amplification cycle of 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 2 minutes, and 72 ° C for 7 minutes. Heated. In a sample of cells that have undergone homologous recombination with the KO3 vector, approximately 1.6 kb of DNA is amplified. This PCR detected cells that had undergone homologous recombination with the KO3 vector, and also confirmed that homologous recombination with the KO2 vector remained.

(9) PCR screening results In Method 1, 616 cells were analyzed, of which 18 cells were considered to be homologous recombinants (homologous recombination efficiency was 2.9%). In Method 2, 524 cells were analyzed, of which 2 cells were considered to be homologous recombinants (the homologous recombination efficiency was about 0.4%). Furthermore, in Method 3, 382 cells were analyzed, of which 7 cells were considered to be homologous recombinants (homologous recombination efficiency was about 1.8%).

(10) Southern blot analysis Analysis was performed according to the method described above. As a result, one cell that was completely deficient in the fucose transporter gene was found. In the first-stage knockout, the PCR and Southern blot analysis results matched, but in the second-stage knockout, they did not.

(11) Expression analysis of fucose Further, the expression of fucose in 26 cells determined to be homologous recombinants by PCR was analyzed. Ice-cold 1 × 10 ⁶ cells with 100 μl of PBS containing 5 μg / ml Lens culinaris Agglutinin, FITC Conjugate (Vector Laboratories), 2.5% FBS, 0.02% sodium azide (hereinafter referred to as FACS lysate) Stained for 1 hour. Thereafter, the cells were washed three times with a FACS lysate and measured with a FACSCalibur (Becton Dickinson). As a result, it was revealed that the expression of fucose was reduced only in the FTP-KO strain, which was a cell determined to be completely deficient in the fucose transporter gene by Southern blot analysis.

[Reference Example 23] Establishment of antibody-producing cells derived from the FTP-KO strain and purification of antibodies produced by the cells
Example 2 In a SFMII (+) medium, the final concentration of hygromycin B was adjusted to 1 mg / ml.
The fucose transporter-deficient strain (FT-KO cells, clone name 3F2) obtained in 1 was passaged. 8 × 10 ⁶ 3F2 were suspended in 0.8 ml Dulbecco's phosphate buffer. 25 μg of humanized glypican 3 antibody expression vector was added to the cell suspension, and the cell suspension was transferred to a Gene Pulser Cuvette. After leaving on ice for 10 minutes, the vector was introduced into the cells by electroporation with GENE-PULSER II under conditions of 1.5 kV and 25 μFD. After introducing the vector, the cells were suspended in 40 ml of SFMII (+) medium, and seeded at 100 μl / well in a 96-well flat bottom plate (Iwaki). After culturing the plate in a CO ₂ incubator for 24 hours at 37 ° C., Geneticin (Invitrogen) was added to a final concentration of 0.5 mg / ml. The antibody production of cells that became resistant to drugs was measured, and humanized glypican 3 antibody-producing cell lines were established.

  The culture supernatant was recovered from the antibody expression strain and applied to a Hitrap rProtein A (Pharmacia) column using a P-1 pump (Pharmacia). The column was washed with a binding buffer (20 mM sodium phosphate (pH 7.0)), and the bound antibody was eluted with an elution buffer (0.1 M Glycin-HCl (pH 2.7)). The eluate was immediately neutralized with a neutralization buffer (1M Tris-HCl (pH 9.0)). After antibody elution fractions were selected and pooled by DC protein assay (BIO-RAD), the elution fractions were concentrated to about 2 ml with Centriprep-YM10 (Millipore). Next, the concentrated solution was subjected to gel filtration using Superdex200 26/60 (Pharmacia) equilibrated with 20 mM acetate buffer (pH 6.0) containing 150 mM NaCl. The peak of the monomer fraction of the eluate was collected, and the fraction was concentrated with Centriprep-YM10. The concentrated solution was filtered using a MILLEX-GW 0.22 μm Filter Unit (Millipore) and stored at 4 ° C. The concentration of the purified antibody was determined by conversion from the molar extinction coefficient based on the absorbance measured at a wavelength of 280 nm.

[Reference Example 24] Analysis of sugar chain binding to humanized anti-glypican 3 antibody produced by FT-KO cells
(1) Preparation of 2-aminobenzamide-labeled sugar chain (2-AB-modified sugar chain) N-Glycosidase F (Roche diagnostics) acts on the FT-KO cell-producing antibody of the present invention and the CHO cell-producing antibody as a control sample. By doing so, the sugar chain binding to the antibody was released from the protein (Weitzhandler M. et al., Journal of Pharmaceutical Sciences (1994) 83 (12) ,, 1670-5). After deproteinization with ethanol (Schenk B. et al., The Journal of Clinical Investigation (2001), 108 (11), 1687-95), the free sugar chain was concentrated to dryness and then 2-aminopyridine (Bigge JC et al., Analytical Biochemistry (1995) 230 (2), 229-238). The obtained 2-AB sugar chain was dereagentd by solid phase extraction using a cellulose cartridge and then concentrated by centrifugation. The purified 2-AB sugar chain was subjected to subsequent analysis as a purified 2-AB sugar chain. Next, β-Galactosidase (Seikagaku Corporation) was allowed to act on the purified 2-AB sugar chain to prepare an agalactosyl 2-AB sugar chain.

(2) Analysis of agaractosyl 2-AB-modified glycan by normal phase HPLC Using the FT-KO cell-producing antibody of the present invention and a glycan released from a CHO cell-producing antibody as a control sample as a starting material. The prepared agalactosyl 2-AB sugar chain was analyzed by normal phase HPLC using an amide column TSKgel Amide-80 (Tosoh), and the chromatograms were compared. In CHO cell-producing antibodies, G (0) is present as the main component of the sugar chain, and G (0) -Fuc without added fucose is 4% of the total sugar chain based on the calculation from the peak area ratio. It was estimated to exist to some extent. On the other hand, in the FT-KO cell-producing antibody, G (0) -Fuc is the main component, and 90% of the total sugar chain is based on the calculation from the peak area ratio in the antibody produced from any production strain. The above was present as a sugar chain to which fucose was not added.

[Example 25] Establishment of stable expression strain of humanized H0L0 antibody and its point mutation-modified antibody Hspu2.2Lspu2.2 (Hu2.2Lu2) which is a modified antibody of H0L0 antibody prepared by the method described in Example 21 .2) The gene encoding the antibody and the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody or the H0L0 antibody subjected to the modification was cloned into an expression vector. Upon cloning, the genes encoding the H and L chains were inserted into separate expression vectors so that the genes encoding the H and L chains constituting the antibody were expressed. FTP-KO prepared in Reference Example 22 was prepared by cleaving two types of expression vectors selected with PvuI so that each gene encoding the H chain and L chain had a desired combination as described above. It was introduced into the strain using electroporation.

The transduction strain that stably produces the H0L0 antibody and its modified antibody by electroporation was produced using Gene PulserII (Bio Rad). 10 μg each of the H chain and L chain expression plasmid DNAs resulting in the combination comprising the desired H chain and L chain and 0.75 ml of CHO cells (1 × 10 ⁷ cells / ml) suspended in PBS are mixed, and the mixture is on ice Left for 10 minutes. After the mixed solution was transferred to the Gene Pulser II cuvette, an electric pulse was applied at a capacity of 1.5 kV and 25 μFD. The pulsed mixture was allowed to stand at room temperature for 10 minutes and then suspended in CHO-S-SFMII / 1% HT / 1% PS medium. In each well of a 96-well culture plate, 100 μl of each diluted suspension prepared in the same medium at 5, 10, and 50 times was dispensed. The plates were incubated for 24 hours in a CO ₂ incubator maintained at 5% CO ₂ concentration. Thereafter, the plate with Geneticin (GIBCO) added to each well to a final concentration of 500 μg / ml and Zeocin (Invitrogen) to a final concentration of 600 μg / ml was further incubated for 2 weeks. Colonies of transduced cells exhibiting geneticin and Zeocin resistance were further selected by passage on the same medium containing 500 μg / ml Geneticin (GIBCO) and 600 μg / ml Zeocin (Invitrogen). By evaluating the antibody concentration in the culture supernatant of the transduced cells selected as described above using BiacoreQ (BIACORE), a transduced strain that highly expresses the desired antibody was established. Measurement of the antibody concentration in the culture supernatant was carried out based on the protocol attached to BiacoreQ (BIACORE).

[Example 26] Drug efficacy test of humanized H0L0 antibody and its point mutation modified antibody using in vivo model
(1) Maintenance of cell lines for transplantation to in vivo models
Hep G2 cells (ATCC) were used. Hep G2 cells are referred to as Minimun Essential Medium Eagle medium (SIGMA) containing 10% FBS, 1 mmol / l MEM Sodium Pyruvate (Invitrogen), and 1 mmol / l MEM Non-Essential Amino Acid (Invitrogen) (hereinafter referred to as passage medium). ) Passaged and maintained.

(2) Preparation of mouse model transplanted with Hep G2 cells
A cell suspension of Hep G2 cells was prepared at 5 × 10 ⁷ cells / ml using a solution containing passage medium and MATRIGEL Matrix (BD Bioscience) at a ratio of 1: 1. On the day before cell transplantation, SCID mice (male, 5 weeks old) were administered intraperitoneally with 100 μl of anti-asialo GM1 antibody (Wako Pure Chemical Industries, 1 vial contents were dissolved in 5 ml of the solution). ) (Japan Claire) was transplanted 100 μl of the cell suspension (5 × 10 ⁶ cells / mouse) subcutaneously into the abdomen. The tumor volume was calculated using the formula: tumor volume = major axis × minor axis × minor axis / 2, and it was judged that the model was established when the average tumor volume reached 130-330 mm ³ .

(3) Preparation of administration sample containing each test antibody
The administration sample containing each antibody of H0L0 antibody, Hu2.2Lu2.2 antibody, and Hd1.8Ld1.6 antibody is 0.5 mg / ml (5 mg / kg administration group) or 0.1 using physiological saline on the day of administration. It was prepared to be mg / ml (1 mg / kg administration group).

(4) Administration of administration sample containing antibody Administration prepared in (3) above, once a week from the 27th day after transplantation of Hep G2 cells to the mouse model prepared in (2), for a period of 3 weeks Samples were administered via the tail vein at a dose of 10 ml / kg. As a negative control, physiological saline was similarly administered from the tail vein once a week for a period of 3 weeks at a dose of 10 ml / kg. In any group, 5 mice were taken as one group, and administration of administration samples containing each test antibody was performed for each group. Almost simultaneously with the administration, venous blood was collected from 3 individuals in each group as a test substance used to measure the blood concentration of each antibody in the mouse. Specifically, blood was collected from the dorsal foot vein at two time points immediately before the second administration, 0.5 hours after the first administration. A 20 μl volume of blood was collected by heparin treatment and plasma was prepared by centrifugation.

(5) Evaluation of anti-tumor effect of each test antibody The anti-tumor effect of each test antibody in a mouse model transplanted with human liver cancer was evaluated by measuring the tumor volume one week after the last day of administration of the administration sample. As a result, as shown in FIG. 55, the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody tends to have a strong drug effect, and the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody tends to have a weak drug effect. was there.

(6) Blood concentration of each test antibody The test antibody concentration in mouse plasma was measured by a method according to the ELISA method described in Example 21. Calibration curve samples with plasma concentrations of 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2 μg / ml were prepared. An immunoplate (Nunc-Immuno Plate, MaxiSoup (Nalge nunc International)) in which a solid Glypican-3 core (manufactured by Chugai Pharmaceutical Co., Ltd.) is immobilized on a calibration curve sample and a mouse plasma test sample appropriately diluted to a desired concentration And the plate was allowed to stand at room temperature for 1 hour. Subsequently, Goat Anti-Human IgG-BIOT (Southern Biotechnology Associates) and Streptavidin-alkaline phosphatase conjugate (Roche Diagnostics) were sequentially dispensed, and a color reaction was performed using BluePhos Microwell Phosphatase Substrates System (Kirkegaard & PerryLaboratories) as a substrate. It was. The color of the reaction solution in each well was calculated by measuring the absorbance at 650 nm of the reaction solution using a microplate reader. Based on the calibration curve created from the absorbance of each calibration curve sample, the antibody concentration in mouse plasma was calculated using analysis software SOFTmax PRO (Molecular Devices).

  Mouse plasma concentrations at 30 minutes and 7 days after administration are shown in FIG. It was shown that the antibody concentration in the plasma of mice 7 days after administration increased when the pI of the test antibody was further decreased at any dose of the test antibody.

[Example 27] ADCC activity of each test antibody using human peripheral blood mononuclear cells as effector cells ADCC of each test antibody using human peripheral blood mononuclear cells (hereinafter referred to as human PBMC) as effector cells Activity was measured as follows.

(1) Preparation of human PBMC solution
50 ml of peripheral blood was obtained from a healthy volunteer (adult male) belonging to Chugai Pharmaceutical Co., Ltd. using a syringe into which 200 μl of a 1000 unit / ml heparin solution (Novo-Heparin injection 5,000 units, Novo Nordisk) was injected in advance. It was collected. Leucosep lymphocyte separation tube (Greiner bio-one) that had been diluted into 4 aliquots with PBS (-), divided into 4 equal parts, and 15 ml Ficoll-Paque PLUS was previously injected and centrifuged. ). After the separation tube into which the peripheral blood was dispensed was centrifuged at a speed of 2150 rpm for 10 minutes at room temperature, the mononuclear cell fraction layer was collected. After the cells contained in each layer are washed once with Dulbecco's Modified Eagle's Medium (SIGMA) containing 10% FBS (hereinafter referred to as 10% FBS / D-MEM), the cells are treated with 10% FBS / D. -Suspended in MEM to a cell density of 5 × 10 ⁶ / ml. The cell suspension was subjected to subsequent experiments as a human PBMC solution.

(2) Preparation of target cells
Hep G2 cells were detached from the dish and seeded on a 96-well U-bottom plate at 1 × 10 ⁴ cells / well. The plates were incubated overnight at 37 ° C. in a 5% carbon dioxide incubator. The next day, 5.55 MBq Cr-51 was added to each well of the plate and the plate was incubated at 37 ° C. for 3 hours in a 5% carbon dioxide incubator. Hep G2 cells present in each well of the plate were used as target cells for the subsequent measurement of ADCC activity.

(3) Chromium release test (ADCC activity)
ADCC activity is evaluated by the specific chromium release rate by the chromium release method. H0L0 antibody, Hu2.2Lu2.2 antibody, Hd1.8Ld1 prepared at each concentration (0, 0.004, 0.04, 0.4, 4, 40 μg / ml) after washing the target cells prepared in (2) 100 μl of each antibody of .6 antibody was added respectively. The plate was reacted at room temperature for 15 minutes, and then the antibody solution was removed. Next, the plate with 100 μl of subculture medium added to each well was incubated at 37 ° C. for 1 hour in a 5% carbon dioxide incubator. The plate to which 100 μl (5 × 10 ⁵ cells / well) of the human PBMC solution prepared in (1) was added in each well was left at 37 ° C. for 4 hours in a 5% carbon dioxide incubator, and then centrifuged. Separation operation was performed. Radioactivity of 100 μl of culture supernatant in each well of the plate was measured using a gamma counter. The following formula: Specific chromium release rate (%) = (AC) x 100 / (BC)
Based on this, the specific chromium release rate was determined.

  In the above formula, A represents the average value of radioactivity (cpm) of 100 μl of the culture supernatant in each well. B shows the radioactivity of 100 μl of the culture supernatant in the wells in which 100 μl of 2% NP-40 aqueous solution (Nonidet P-40, Nacalai Tesque) and 50 μl of 10% FBS / D-MEM medium were added to the target cells ( cpm). Further, C represents an average value of radioactivity (cpm) of 100 μl of the culture supernatant in a well obtained by adding 150 μl of 10% FBS / D-MEM medium to the target cells. The test was performed in triplicate, and the average value and standard deviation of the specific chromium release rate (%) in the test reflecting the ADCC activity of each test antibody were calculated.

(4) Evaluation of ADCC activity of each test antibody As a result of evaluating ADCC activity exhibited by human PBMC via each test antibody, it was confirmed that all test antibodies had ADCC activity. The result is shown in FIG. As a result of performing a significant difference test on the specific chromium release rate exhibited by each test antibody at each concentration, the specific chromium release rate exhibited by each test antibody at all antibody concentrations was significantly different between the test antibodies. There was no difference. The SAS preclinical package (SAS Institute Inc.) was used for statistical analysis. Based on the above results, it was shown that there was no difference between ADCC activity of each test antibody whose pI was modified.

[Example 28] Production of anti-human IL-6 receptor antibody, anti-human GPC3 antibody, anti-human IL-31 receptor antibody
1. Preparation of anti-human IL-6 receptor antibody Two types of antibodies were prepared as anti-human IL-6 receptor antibodies. 6R_a_H1 (SEQ ID NO: 229) composed of 6R_a_H1 (SEQ ID NO: 221) as the H chain, 6R_a_H1L1 composed of 6R_a_L1 (SEQ ID NO: 224) as the L chain, and 6R_b_H1 (SEQ ID NO: 227) as the H chain. 6R_b_H1L1 composed of According to Reference Examples 1 and 2, animal cell expression vectors encoding the respective amino acid sequences were prepared, and expressed and purified.

2 Preparation of anti -human GPC3 antibody An anti-human GPC3 antibody was prepared. GPC3_H1L1 composed of GPC3_H1 (SEQ ID NO: 233) as the H chain and GPC3_L1 (SEQ ID NO: 236) as the L chain was prepared. According to Reference Examples 1 and 2, animal cell expression vectors encoding the respective amino acid sequences were prepared, and expressed and purified.

3 Anti-human IL-31 receptor antibody An anti-human IL-31 receptor antibody was prepared. 31R_H1L1 composed of 31R_H1 (SEQ ID NO: 239) as the H chain and 31R_L1 (SEQ ID NO: 242) as the L chain was prepared. According to Reference Examples 1 and 2, animal cell expression vectors encoding the respective amino acid sequences were prepared, and expressed and purified.

[Example 29] Reduction of isoelectric point by amino acid substitution of anti-human IL-6 receptor antibody, anti-human GPC3 antibody, anti-human IL-31 receptor antibody
1 Search for CDR sequences that reduce the isoelectric point without diminishing the antigen binding activity
In WO / 2007/114319, an example of controlling the isoelectric point by amino acid substitution in CDR is shown, but amino acid substitution is performed on H chain CDR3, and H chain CDR3 is greatly involved in the binding activity of the antibody to the antigen. Therefore, depending on the type of antibody, it was expected that the isoelectric point could not be lowered without reducing the binding activity to the antigen by amino acid substitution at the same position. Therefore, a candidate CDR sequence that can lower the isoelectric point without diminishing the antigen-binding activity regardless of the type of antibody was searched. As a result, in the H chain variable region, H31, H52, H61, H62, H64, and H65 are converted to the antigen, and in the L chain variable region, L24, L27, L27a, L53, L54, L55, and L56 (Kabat numbering) are converted into the antigen. It was mentioned as a candidate CDR sequence capable of lowering the isoelectric point without diminishing the binding activity of. Therefore, in the following anti-human IL-6 receptor antibody, anti-human GPC3 antibody, and anti-human IL-31 receptor antibody, amino acid substitution was performed for some of these candidate CDR sequences, and antigen-binding activity was determined. We examined whether it is possible to reduce the isoelectric point without attenuating.

2 Preparation of anti-human IL-6 receptor antibody with reduced isoelectric point, evaluation of binding activity, and 6R_a_H1 (SEQ ID NO: 221) and 6R_a_L1 constituting 6R_a_H1L1, which is an anti-human IL-6 receptor antibody with isoelectric point measurement 6R_a_H2 (SEQ ID NO: 222) and 6R_a_L2 (SEQ ID NO: 225) into which amino acid substitution that lowers the isoelectric point and other amino acid substitutions were introduced respectively (SEQ ID NO: 224) According to the method, a vector was prepared, and 6R_a_H2L2 was expressed and purified. A vector was prepared from 6R_a_H3 (SEQ ID NO: 223) and 6R_a_L3 (SEQ ID NO: 226) into which 6R_a_H2L2 was further introduced with an amino acid substitution that lowers the isoelectric point and other amino acid substitutions according to the method of Reference Example 1. 6R_a_H3L3 was expressed and purified.
The dissociation constant (KD) for human IL-6 receptor, which is an antigen of 6R_a_H1L1, 6R_a_H2L2, and 6R_a_H3L3, was measured using the Biacore T100 method described in Reference Example 3. The dissociation constants (KD) of 6R_a_H1L1, 6R_a_H2L2, and 6R_a_H3L3 with respect to the IL-6 receptor were the same as shown in Table 18 below, and no significant reduction in antigen-binding activity was observed due to the introduction of amino acid substitutions.

  As a result of measuring the isoelectric point by isoelectric focusing known to those skilled in the art, the isoelectric point of 6R_a_H1L1 was about 9.2, whereas the isoelectric point of 6R_a_H2L2 in which amino acid substitution was performed to reduce the isoelectric point Is about 6.1, and the isoelectric point of 6R_a_H3L3 is about 5.4, which is about 3.1 and 3.8 lower than 6R_a_H1L1, respectively. Moreover, when the theoretical isoelectric point of variable region VH / VL was calculated by GENETYX (GENETYX CORPORATION), the theoretical isoelectric point of 6R_a_H1L1 was 9.37, whereas the theoretical isoelectric point of 6R_a_H2L2 was 4.63, The theoretical isoelectric point of 6R_a_H3L3 was about 4.27, and the theoretical isoelectric point decreased by 4.74 and 5.10 compared to 6R_a_H1L1, respectively. These results are summarized in Table 19.

  Table 20 below summarizes the amino acid substitutions of the CDR sequences introduced for 6R_a_H1L1. These CDR amino acid substitutions can reduce the isoelectric point of antibody molecules without significantly reducing the antigen-binding activity against 6R_a_H1L1, an anti-human IL-6 receptor antibody. I found it.

Subsequently, with respect to 6R_b_H1 (SEQ ID NO: 227) and 6R_b_L1 (SEQ ID NO: 229) constituting 6R_b_H1L1, which is another anti-human IL-6 receptor antibody, amino acid substitution and other amino acids that reduce the isoelectric point, respectively. A vector was prepared from 6R_b_H2 (SEQ ID NO: 228) and 6R_b_L2 (SEQ ID NO: 230) into which substitution was introduced according to the methods of Reference Examples 1 and 2, and 6R_b_H2L2 was expressed and purified. A vector was prepared from 6R_b_L3 (SEQ ID NO: 231) and 6R_b_L4 (SEQ ID NO: 232) into which 6R_b_H2L2 was further introduced with an amino acid substitution that lowers the isoelectric point and other amino acid substitutions according to the method of Reference Example 1. 6R_b_H2L3 and 6R_b_H2L4 were expressed and purified.
The neutralizing activity of 6R_b_H1L1, 6R_b_H2L2, 6R_a_H2L3, 6R_b_H2L4, which is an antigen of human IL-6 receptor, was measured by the method shown in Reference Example 4. The neutralization activities of 6R_b_H1L1, 6R_b_H2L2, 6R_a_H2L3, and 6R_b_H2L4 were almost the same as shown in FIG. 58, and no significant reduction in antigen-binding activity was observed due to the introduction of amino acid substitution.

  As a result of measuring the isoelectric point by isoelectric focusing known to those skilled in the art, the isoelectric point of 6R_b_H1L1 was about 9.3, whereas the isoelectric point of 6R_b_H2L2 in which amino acid substitution was performed to reduce the isoelectric point Was about 5.9, and the isoelectric point decreased by about 3.4 compared to 6R_b_H1L1. Further, when the theoretical isoelectric point of variable region VH / VL was calculated by GENETYX (GENETYX CORPORATION), the theoretical isoelectric point of 6R_b_H1L1 was 9.20, whereas the theoretical isoelectric point of 6R_b_H2L2 was 4.52. The theoretical isoelectric point of 6R_b_H2L3 is about 4.46, the theoretical isoelectric point of 6R_b_H2L4 is about 4.37, and the theoretical isoelectric point is decreased by 4.68, 4.74, and 4.83, respectively, compared with 6R_b_H1L1. These results are summarized in Table 21.

  Table 22 below summarizes the amino acid substitutions of the CDR sequences introduced for 6R_b_H1L1. These CDR amino acid substitutions can reduce the isoelectric point of antibody molecules without significantly reducing the binding activity to the antigen against 6R_b_H1L1, an anti-human IL-6 receptor antibody. I found it.

3. Preparation of anti-human GPC3 antibody with reduced isoelectric point, evaluation of binding activity, and GPC3_H1 (SEQ ID NO: 233) and GPC3_L1 (SEQ ID NO: 236) constituting anti-human GPC3 antibody GPC3_H1L1 In contrast, GPC3_H2 (SEQ ID NO: 234) and GPC3_L2 (SEQ ID NO: 237) into which amino acid substitutions that lower the isoelectric point and other amino acid substitutions were introduced were prepared according to the methods of Reference Examples 1 and 2, respectively. Then, GPC3_H2L2 was expressed and purified. A vector was prepared from GPC3_H3 (SEQ ID NO: 235) and GPC3_L3 (SEQ ID NO: 238) into which GPC3_H2L2 was further introduced with an amino acid substitution that lowers the isoelectric point and other amino acid substitutions according to the method of Reference Example 1. GPC3_H3L3 was expressed and purified.
The binding activity of GPC3_H1L1, GPC3_H2L2, and GPC3_H3L3 to human GPC3, which is an antigen, was evaluated using the competitive ELISA method described in Reference Example 5, and the results are shown in FIGS. 59 and 60. The binding activities of GPC3-H1L1 and GPC3-H2L2 and GPC3-H2L2 and GPC3-H3L3 to glypican 3 were almost the same, and no significant decrease in the binding activity to the antigen due to the introduction of amino acid substitution was observed.

  As a result of measuring the isoelectric point by isoelectric focusing known to those skilled in the art, the isoelectric point of GPC3_H1L1 was about 9.6, whereas the isoelectric point of GPC3_H2L2 with amino acid substitution to lower the isoelectric point was Was about 8.9, and the isoelectric point of GPC3_H2L2 was 0.7 lower than that of GPC3_H1L1. Similarly, the isoelectric point of GPC3_H2L2 was about 8.7, whereas the isoelectric point of GPC3_H3L3 with amino acid substitution to reduce the isoelectric point was about 6.5, and the isoelectric point of GPC3_H3L3 was the isoelectric point of GPC3_H2L2 Compared to the point, it decreased by 2.2. The theoretical isoelectric point of GPC3_H1L1 was 9.65, whereas the theoretical isoelectric point of GPC3_H2L2 was 8.47, and the theoretical isoelectric point of GPC3_H2L2 was 1.18 lower than that of GPC3_H1L1. Similarly, the theoretical isoelectric point of GPC3_H2L2 was 8.47, whereas the theoretical isoelectric point of GPC3_H3L3 was 4.93, and the theoretical isoelectric point of GPC3_H2L2 was 3.54 lower than that of GPC3_H1L1. These results are summarized in Table 23.

  Table 24 below summarizes the amino acid substitutions of the CDR sequences introduced for GPC3_H1L1. It has been found that the amino acid substitution of these CDRs can lower the isoelectric point of the antibody molecule without significantly reducing the binding activity to the antigen against GPC3_H1L1, which is an anti-human GPC3 antibody.

4 Anti-human IL-31 receptor antibody with reduced isoelectric point, binding activity evaluation, and isoelectric point measurement
31R_H2 (SEQ ID NO: 240) and 31R_L2 (SEQ ID NO: 240) and 31R_H2 (SEQ ID NO: 240) and 31R_H2 (SEQ ID NO: 239) and 31R_L1 (SEQ ID NO: 242) constituting 31R_H1L1 were introduced with amino acid substitutions that lower the isoelectric point and other amino acid substitutions, respectively. A vector was prepared from SEQ ID NO: 243) according to the methods of Reference Examples 1 and 2, and 31R_H2L2 was expressed and purified. 31R_H2L2 was further introduced into 31R_H3 (SEQ ID NO: 241) introduced with amino acid substitution that lowers the isoelectric point and other amino acid substitutions according to the method of Reference Example 1, and 31R_H3L2 was expressed and purified. It was.
The affinity of 31R_H2L2 and 31R_H3L2 to IL-31 was evaluated using the method by Biacore described in Reference Example 6, and the results are summarized in Table 25. As shown in Table 25, 31R_H2L2 and 31R_H3L2 have almost the same binding activity of 31R_H1L1 to NR10, and no significant reduction in binding activity to the antigen due to the introduction of amino acid substitution was observed.

  As a result of measuring the isoelectric point by isoelectric focusing known to those skilled in the art, the isoelectric point of 31R_H1L1 was about 7.76, whereas the isoelectric point of 31R_H2L2 in which amino acid substitution was performed to reduce the isoelectric point was performed. The point was about 5.49, and the isoelectric point of 31R_H3L2 was about 5.43, which was about 2.27 and about 2.33 lower than that of 31R_H1L1, respectively. The theoretical isoelectric point of 31R _H1L1 was 7.76, whereas the theoretical isoelectric point of 31R _H2L2 was 4.63, and the theoretical isoelectric point of 31R _H3L2 was about 4.54, compared with 31R _H1L1 respectively. The theoretical isoelectric point decreased by about 3.13 and about 3.22. These results are summarized in Table 26.

  Table 27 below summarizes the amino acid substitutions of the CDR sequences introduced for 31R_H1L1. These CDR amino acid substitutions can reduce the isoelectric point of antibody molecules without significantly reducing the antigen-binding activity against 31R_H1L1, an anti-human IL-31 receptor antibody. I found it.

5 CDR sequences that could reduce the isoelectric point of anti-human IL-6 receptor antibody, anti-human GPC3 antibody, and anti-human IL-31 receptor antibody without diminishing the antigen-binding activity. Table 28 shows the H chain CDR sequences and Table 29 shows the L chain CDR sequences of anti-human IL-6 receptor antibodies (6R_a and 6R_b), anti-human GPC3 antibody (GPC3), and anti-human IL-31 receptor antibody (31R). Summarized. Amino acid substitutions that were able to lower the isoelectric point without diminishing the antigen binding activity were filled in and displayed.

  Thus, H31, H61, H62, H64, H65 in the H chain variable region, and L24, L27, L53, L54, and L55 (Kabat numbering) in the L chain variable region are not dependent on the type of antibody, and a plurality of antibodies Was found to be a common CDR site that can introduce amino acid substitutions that lower the isoelectric point of the antibody without significantly diminishing the antibody's binding activity to the antigen.

  WO / 2007/114319 shows that IgG pharmacokinetics can be improved by lowering the isoelectric point of an antibody. In WO / 2007/114319, in order not to reduce the binding activity to the antigen, amino acid substitutions are mainly performed on the framework of the antibody variable region. About anti-FactorIXa antibody, about 0.9 change as the measured isoelectric point, theory The isoelectric point was about 1.0 change, the anti-FactorX antibody was about 0.5 change as the measured isoelectric point, and the theoretical isoelectric point was about 0.1 change, and the degree of change of the isoelectric point was small. .

  In the present invention, it has become possible to find a CDR sequence that does not diminish the antigen-binding activity, and to introduce an amino acid substitution that lowers the isoelectric point not only in the framework of the antibody variable region but also in the CDR. Thereby, in the above-mentioned anti-human IL-6 receptor antibody, a decrease of about 3.8 as the measured isoelectric point and a decrease of about 5.1 as the theoretical isoelectric point are achieved, and in the anti-human GPC3 antibody, the measured isoelectric point As a result, the theoretical isoelectric point was reduced by about 3.1, and the theoretical isoelectric point was reduced by about 4.7. In the anti-human IL-31 receptor antibody, the measured isoelectric point was reduced by about 3.2, and the theoretical isoelectric point was reduced by about 2.3. As a result, it was found that a significant reduction in isoelectric point can be achieved as compared with amino acid substitution only for the framework.

[Example 30] Evaluation of pharmacokinetics of anti-human IL-6 receptor antibody, anti-human GPC3 antibody and anti-human IL-31 receptor antibody with reduced isoelectric point
1. Evaluation of pharmacokinetics of anti-human IL-6 receptor antibody in cynomolgus monkeys and mice Cynomolgus monkeys of 6R_a_H1L1, an anti-human IL-6 receptor antibody, and 6R_a_H2L2 and 6R_a_H3L3, anti-human IL-6 receptor antibodies with reduced isoelectric point The pharmacokinetics in were evaluated. 6R_a_H1L1 and 6R_a_H2L2 were each administered intravenously at 1.0 mg / kg, and blood was collected over time before and after administration. In addition, 6R_a_H2L2 and 6R_a_H3L3 were each administered subcutaneously at a dose of 1.0 mg / kg, and blood was collected over time before and after administration.

  Plasma concentration was measured by ELISA. An immunoplate (Nunc-Immuno Plate, MaxiSorp (Nalge nunc) with a calibration curve sample and plasma measurement sample of appropriate concentration immobilized on Anti-human IgG (γ-chain specific) F (ab ') 2 (Sigma) International)) and allowed to stand at room temperature for 1 hour, and then sequentially reacted with Goat Anti-Human IgG-BIOT (Southern Biotechnology Associates) and Streptavidin-alkaline phosphatase conjugate (Roche Diagnostics). A color reaction was performed using Microwell Phosphatase Substrates System (Kirkegaard & Perry Laboratories) as a substrate, and the absorbance at 650 nm was measured with a microplate reader. The plasma concentration was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices). The obtained plasma concentration transition data were subjected to non-model-dependent analysis using pharmacokinetic analysis software WinNonlin (manufactured by Pharsight), and clearance (CL) was calculated and shown in Table 30. When 6R_a_H1L1 and 6R_a_H2L2 administered intravenously were compared, it was confirmed that 6R_a_H2L2, which had a reduced isoelectric point, had a small clearance and improved pharmacokinetics by reducing the isoelectric point. Furthermore, when 6R_a_H2L2 and 6R_a_H3L3 administered subcutaneously were compared, it was confirmed that 6R_a_H3L3, which had a reduced isoelectric point, had a small clearance and improved pharmacokinetics by lowering the isoelectric point.

Subsequently, the pharmacokinetics of 6R_b_H1L1, which is a different anti-human IL-6 receptor antibody, and 6R_b_H2L2, which is an anti-human IL-6 receptor antibody with reduced isoelectric point (C57BL / 6J, Charles River, Japan), were evaluated. 6R_b_H1L1 and 6R_b_H2L2 were each administered intravenously at 1.0 mg / kg, and blood was collected over time before and after administration. In addition, 6R_b_H1L1 and 6R_b_H2L2 were each subcutaneously administered at 1.0 mg / kg, and blood was collected over time before and after administration.
Plasma concentration was measured by ELISA. First, Recombinant Human IL-6 sR (R & D Systems) was biotinized using EZ-Link ™ Sulfo-NFS-Biotinylation Kit (PIERCE). Distribute this biotinylated human-sIL-6R to Reacti-Bind Streptavidin High Binding Capacity (HBC) Coated Plates (manufactured by PIERCE) and let it stand at room temperature for 1 hour or more to create a human-sIL-6R solid-phase plate did. A calibration curve sample and a mouse plasma measurement sample having appropriate concentrations were prepared, dispensed onto a human-sIL-6R solid-phased plate, and allowed to stand at room temperature for 1 hour. Anti-human IgG-AP (manufactured by SIGMA) was then reacted, color reaction was performed using BluePhos Microwell Phosphatase Substrates System (Kirkegaard & Perry Laboratories) as a substrate, and absorbance at 650 nm was measured with a microplate reader. . The plasma concentration was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices). Table 31 shows the clearance (CL) calculated by non-model-dependent analysis of the obtained plasma concentration transition data using pharmacokinetic analysis software WinNonlin (Pharsight). When 6R_a_H1L1 and 6R_a_H2L2 administered intravenously and subcutaneously were compared, it was confirmed that 6R_a_H2L2, which had a reduced isoelectric point, had a small clearance and improved pharmacokinetics by reducing the isoelectric point.

2 Evaluation of pharmacokinetics of anti-human GPC3 antibody in mice Anti-human GPC3 antibody GPC3_H1L1, and anti-human GPC3 antibodies GPC3_H2L2 and GPC3_H3L3 with reduced isoelectric point were analyzed in CB-17 / Icr-scid mice. evaluated. GPC3_H1L1, GPC3_H2L2, and GPC3_H3L3 were each administered once intravenously at 5.0 mg / kg, and blood was collected over time before and after administration.
Plasma concentration was measured by ELISA. A calibration curve sample of an appropriate concentration and a mouse plasma test sample appropriately diluted to a desired concentration are immunoplates on which GPC3 (manufactured by Chugai Pharmaceutical Co., Ltd.) as an antigen is immobilized (Nunc-Immuno Plate, MaxiSoup (Nalge nunc International)), and the plate was allowed to stand at room temperature for 1 hour. Subsequently, Goat Anti-Human IgG-BIOT (Southern Biotechnology Associates) and Streptavidin-alkaline phosphatase conjugate (Roche Diagnostics) were sequentially dispensed, and a color reaction was performed using BluePhos Microwell Phosphatase Substrates System (Kirkegaard & Perry Laboratories) as a substrate. The absorbance at 650 nm was measured with a microplate reader. The plasma concentration was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices). Table 32 shows the clearance (CL) calculated by non-model-dependent analysis of the obtained plasma concentration transition data using pharmacokinetic analysis software WinNonlin (Pharsight). When GPC3_H1L1 and GPC3_H2L2 are compared, GPC3_H2L2 with a lower isoelectric point has a smaller clearance, and when GPC3_H2L2 and GPC3_H3L3 are compared, GPC3_H3L3 with a lower isoelectric point has a smaller clearance and lowers the isoelectric point. It was confirmed that the pharmacokinetics improved.

3 Evaluation of pharmacokinetics of anti-human IL-31 receptor antibody in mice Anti-human IL-31 receptor antibody 31R_H1L1 and anti-human IL-31 receptor antibody 31R_H2L2 with reduced isoelectric point (C57BL / 6J, Nippon Charles River). 31R_H1L1 and 31R_H2L2 were each administered intravenously at 1.0 mg / kg, and blood was collected over time before and after administration.

  Plasma concentration was measured by ELISA. An immunoplate (Nunc-Immuno Plate, MaxiSorp (manufactured by Nalge nunc International)) on which a calibration curve sample and a plasma measurement sample of an appropriate concentration were immobilized with an anti-human IgG (Fc-specific) antibody (manufactured by Sigma) The solution was dispensed and allowed to stand at room temperature for 1 hour. After reacting Goat Anti-Human IgG-ALP (manufactured by Sigma) for 1 hour at room temperature, a color reaction was performed using BluePhos Microwell Phosphatase Substrates System (manufactured by Kirkegaard & Perry Laboratories) as a substrate, and 650 using a microplate reader. The absorbance at nm was measured. The plasma concentration was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices).

  The obtained plasma concentration transition data were subjected to non-model-dependent analysis using pharmacokinetic analysis software WinNonlin (Pharsight), and clearance (CL) was calculated and shown in Table 33. When 31R_H1L1 and 31R_H2L2 were compared, it was confirmed that 31R_H2L2, which lowered the isoelectric point, had a small clearance, and the disappearance rate was reduced by lowering the isoelectric point.

3 Conclusion In the present invention, we reduce the isoelectric point of an antibody without reducing the binding activity of the antibody to the antigen by amino acid substitution of the CDR sequence in a plurality of antibodies of different types of antigens. It was found that the dynamics can be improved. Among the amino acid substitutions in the found CDR sequences, H31, H61, H62, H64, H65 in the H chain variable region, and L24, L27, L53, L54, L55 (Kabat numbering) in the L chain variable region Amino acid of CDR sequence that can improve pharmacokinetics by introducing amino acid substitution that lowers the isoelectric point of the antibody without significantly reducing the binding activity of the antibody to the antigen in multiple antibodies without depending on It was found to be a substitution. These mutation sites in the CDR sequence are considered to be able to lower the isoelectric point of the antibody without significantly reducing the binding activity of the antibody to the antigen, regardless of the type of antigen. It was considered useful as an amino acid substitution site for improving pharmacokinetics.

[Example 31] Standard chromatographic peak separation of heterodimer and homodimer of anti-human IL-6 receptor antibody, anti-human GPC3 antibody and anti-human IL-31 receptor antibody with reduced isoelectric point
1. Expression of Heterodimer of Anti-Factor IX Antibody / Anti-Factor X Antibody Patent Document WO / 2007/114325 reports a method for purifying an IgG type bispecific antibody having a common L chain. In order to express an IgG type bispecific antibody having a common L chain, it is necessary to express two types of H chains (A chain and B chain) and a common L chain. At this time, not only the target bispecific antibody A chain B chain heterodimer but also the A chain homodimer and the B chain homodimer are expressed, and the target bispecific antibody A is obtained from a mixture of three types of antibodies. It is necessary to purify the chain B chain heterodimer. In this patent, it was impossible to purify the A chain B chain heterodimer by peak separation of the A chain B chain heterodimer, the A chain homodimer and the B chain homodimer by standard chromatography in the conventional method. Is standard chromatography by introducing the difference in isoelectric point between the A chain homodimer and the B chain homodimer by substituting the amino acids in the variable regions of the A chain and B chain, which are two types of H chains. It has been shown that it is now possible to separate the A chain B chain heterodimer, the A chain homodimer and the B chain homodimer by cation exchange chromatography, and to purify the A chain B chain heterodimer. In this patent, as amino acids in the variable region, it was thought that the amino acid substitution of CDR affected the binding activity of the antibody to the antigen, so only the amino acid substitution of the framework was performed. However, as described above, the amino acid substitution of the framework cannot introduce a significant isoelectric point change, and in order to purify the A chain B chain heterodimer more efficiently, the A chain homodimer and the B chain It is desirable to introduce a large isoelectric point difference by homodimer. Thus, by introducing an amino acid substitution in the CDR that lowers the isoelectric point of the antibody without significantly reducing the binding activity of the antibody found in Example 28 to the antigen, A chain B chain heterodimer, A It was investigated whether or not it was possible to separate the chain homodimer and the B chain homodimer.

2 Heterodimer expression of anti-human IL-6 receptor antibody / pI-reduced anti-human IL-6 receptor antibody
6R_a_H1 (SEQ ID NO: 221) as the A chain, 6R_a_H3 (SEQ ID NO: 223) with the isoelectric point lowered and the difference between the A chain and the isoelectric point introduced without decreasing the binding activity to the antigen as the B chain, and Using 6R_a_L3 (SEQ ID NO: 226) as a common L chain, 6R_a_H1H3L3 (A chain B chain heterodimer (6R_a_H1 / H3 / L3), A chain homodimer (6R_a_H1 / L3) and B Expression and purification of chain homodimer (mixture of 6R_a_H3 / L3) was performed.

3. Expression of heterodimer of anti-human GPC3 antibody / pI-reduced anti-human GPC3 antibody
GPC3_H2 (SEQ ID NO: 234) as the A chain, GPC3_H3 (SEQ ID NO: 235) with the isoelectric point lowered and the difference between the A chain and the isoelectric point introduced without reducing the binding activity to the antigen as the B chain, and Using GPC3_L3 (SEQ ID NO: 238) as a common L chain, GPC3_H2H3L3 (A chain B chain heterodimer (GPC3_H2 / H3 / L3), A chain homodimer (GPC3_H2 / L3) and B Expression and purification of the chain homodimer (GPC3_H3 / L3) mixture was performed.

4. Expression of heterodimer of anti-human IL-31 receptor antibody / pI-reduced anti-human IL-31 receptor antibody
31R_H1a (SEQ ID NO: 244) in which the constant region of 31R_H1 was changed as the A chain, and the isoelectric point was lowered and the difference between the A chain and the isoelectric point was introduced as the B chain without decreasing the binding activity to the antigen. 31R_H1aH2aL2 (A chain B chain heterodimer (31R_H1a) was obtained by the method shown in Reference Example 2 using 31R_H2a (SEQ ID NO: 245) in which the constant region was similarly changed and 31R_L2 (SEQ ID NO: 243) as the common L chain. / H2a / L2), A chain homodimer (31R_H1a / L2) and B chain homodimer (31R_H2a / L2) mixture).

6R_a_H1H3L3、GPC3_H2H3L3、および、31R_H1aH2aL2それぞれのA鎖B鎖ヘテロダイマー、A鎖ホモダイマーおよびB鎖ホモダイマーが、陽イオン交換クロマトグラフィーによりピーク分離可能であるかどうかを評価した。標準的な陽イオン交換クロマトグラフィーのカラムとしてProPac WCX-10 (Dionex)を用い、移動相Ａとして25 MES, pH5.0、移動相Ｂとして25 mM MES, 1 M NaCl, pH5.0を使用し、適切な流量およびグラジエントを用いて実施した。陽イオン交換クロマトグラフィーによる評価を行った結果を図６１に示した。その結果、6R_a_H1H3L3、GPC3_H2H3L3、および、31R_H1aH2aL2のいずれにおいても、A鎖B鎖ヘテロダイマー、A鎖ホモダイマーおよびB鎖ホモダイマーのピークが分離することが見出された。 5 Evaluation of Expressed Antibody by Cation Exchange Chromatography Differences in the theoretical isoelectric point of VH / VL between the A chain homodimer and B chain homodimer of the antibody prepared above are summarized in Table 34 below. By introducing an amino acid substitution that reduces the isoelectric point while retaining binding activity, not only in the H chain framework but also in the CDR sequence, a difference of up to 1.56 in the theoretical isoelectric point of the B chain homodimer from the A chain homodimer It became possible to introduce. In patent document WO / 2007/114325, the isoelectric point is decreased by introducing amino acid substitution only into the framework of the A chain homodimer, and the isoelectric point is increased by introducing amino acid substitution only into the framework of the B chain homodimer. It is shown that the difference of 1.13 can be introduced into the theoretical isoelectric point of VH / VL between the A chain homodimer and the B chain homodimer. In this study, the amino acid substitution was performed only on one strand (reduced isoelectric point), but the amino acid substitution was introduced not only in the framework but also in the CDR sequence. It was shown that it is possible to introduce a maximum difference of 1.56. That is, as an amino acid substitution for separating the A chain homodimer and the B chain homodimer, by introducing an amino acid substitution not only in the framework but also in the CDR sequence while retaining the binding activity, the difference in isoelectric point between them can be further increased. It was shown that it is possible to increase. In general, separation by standard ion exchange chromatography depends on the difference in isoelectric point of two components to be separated, and it was considered that this makes it possible to separate the two more easily.

It was evaluated whether the A chain B chain heterodimer, the A chain homodimer, and the B chain homodimer of 6R_a_H1H3L3, GPC3_H2H3L3, and 31R_H1aH2aL2 can be separated by cation exchange chromatography. ProPac WCX-10 (Dionex) is used as a standard cation exchange chromatography column, mobile phase A is 25 MES, pH 5.0, mobile phase B is 25 mM MES, 1 M NaCl, pH 5.0. Was carried out using the appropriate flow rate and gradient. The results of evaluation by cation exchange chromatography are shown in FIG. As a result, it was found that in any of 6R_a_H1H3L3, GPC3_H2H3L3, and 31R_H1aH2aL2, the peaks of the A chain B chain heterodimer, the A chain homodimer and the B chain homodimer were separated.

  In the heavy chain variable region, H31, H61, H62, H64, and H65 (Kabat numbering) do not depend on the type of antibody, and the amino acid substitution at the same position does not significantly reduce the binding activity of the antibody to the antigen. It has been found that the isoelectric point can be lowered, thereby allowing separation of heterodimers and homodimers by cation exchange chromatography. These mutation sites in the CDR sequence are considered to be capable of lowering the isoelectric point of the antibody without significantly reducing the binding activity of the antibody to the antigen, regardless of the type of antigen. It is useful as an amino acid substitution site for increasing the difference in isoelectric point between a heterodimer and a homodimer of a specific antibody.

[Reference Example 1]
Production of Antibody Expression Vector Gene Each mutant was produced by performing QuikChange Site-Directed Mutagenesis Kit (Stratagene) or Assemble PCR using PCR. When QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used, a mutant was prepared by the method described in the attached manual. The method using Assemble PCR was performed using any of the following methods. The first method synthesizes an oligo DNA designed based on the normal and reverse strand sequences including the modification site. A reverse-strand oligo DNA that binds to a normal-chain oligo DNA containing the modification site and a vector into which the gene to be modified is inserted, or a reverse-strand oligo DNA that contains the modification site and a vector into which the gene to be modified is inserted By combining the respective normal-chain oligo DNAs to be combined and performing PCR using PrimeSTAR (TAKARA), two fragments containing the modified site were prepared on the 5-terminal side and the 3-terminal side. Each mutant was generated by joining the two fragments by Assemble PCR. In the two methods, an appropriate number of oligo DNAs were prepared so as to cover the entire variable region, and the entire variable region was prepared by joining these oligo DNAs using Assemble PCR. The mutant produced by these methods was inserted into an expression vector that enables expression of the inserted gene in animal cells, and the base sequence of the obtained expression vector was determined by a method known to those skilled in the art.

[Reference Example 2]
Antibody expression and purified antibody expression were carried out using the following methods. HEK293H strain (Invitrogen) derived from human fetal kidney cancer cells is suspended in DMEM medium (Invitrogen) containing 10% Fetal Bovine Serum (Invitrogen), and the dish (diameter) is used at a cell density of 5-6 × 10 ⁵ cells / mL. 10 mL to each dish of 10 cm, CORNING) After culturing overnight in a CO ₂ incubator (37 ° C, 5% CO ₂ ), the medium is removed by suction and CHO-S-SFM-II (Invitrogen) medium 6.9 mL was added. The prepared plasmid was introduced into cells by the lipofection method. After collecting the obtained culture supernatant, the cells are removed by centrifugation (approximately 2000 g, 5 minutes, room temperature), and further sterilized through a 0.22 μm filter MILLEX (R) -GV (Millipore). Got. The obtained culture supernatant was purified by a method known to those skilled in the art using rProtein A Sepharose ™ Fast Flow (Amersham Biosciences). The purified antibody concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer. The antibody concentration was calculated from the obtained value using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).

[Reference Example 3]
Method for evaluating the affinity of anti-human IL-6 receptor antibody for IL-6 receptor by Biacore
1. Preparation of soluble human IL-6 receptor Recombinant human IL-6 receptor of human IL-6 receptor, which is an antigen, was prepared as follows. J. Biochem. 108, 673-676 (1990) reported soluble human IL-6 receptor consisting of the amino acid sequence from the 1st to 344th N-terminal side (Yamasaki et al., Science 1988; 241: 825-828 (GenBank # X12830)) CHO cell constant expression strain was prepared. From the culture supernatant obtained from soluble human IL-6 receptor-expressing CHO cells, Blue Sepharose 6 FF column chromatography, affinity chromatography using a column immobilized with a specific antibody against soluble human IL-6 receptor, gel filtration Soluble human IL-6 receptor was purified by three column chromatography column chromatography. The fraction eluted as the main peak was used as the final purified product.

2. Evaluation of affinity for soluble human IL-6 receptor by Biacore
Using Biacore T100 (GE Healthcare Bioscience), kinetic analysis of antigen-antibody reaction between anti-human IL-6 receptor antibody and soluble human IL-6 receptor was performed. Rec-Protein A (ZYMED) (hereinafter referred to as Protein A) is immobilized on the sensor chip by a method known to those skilled in the art, the antibody is captured on this immobilized Protein A, and the antigen is reacted as an analyte to react with the antibody. Antigen interaction was measured. The running buffer was HBS-EP +, and the flow rate was 20 μL / min. Each antibody was prepared to bind approximately 100 RU to Protein A / G. The soluble human IL-6 receptor used as the analyte was adjusted to 0, 0.065, 0.131 and 0.261 μg / mL using HBS-EP +. First, the antibody solution was bound to Protein A / G, the analyte solution was allowed to interact there for 3 minutes, then switched to HBS-EP + and the dissociation phase was measured for 10 or 15 minutes. After measurement of the dissociated phase, the sensor chip was regenerated by washing with 10 μL of 10 mM glycine-HCl (pH 1.5). This binding / dissociation / regeneration was defined as one cycle of analysis. Various antibodies were measured according to this cycle. The obtained sensorgrams were analyzed kinetically using Biacore T100 Evaluation Software (GE Healthcare Bioscience), Biacore's exclusive data analysis software.

[Reference Example 4]
Method for evaluating IL-6 receptor neutralizing activity of anti-human IL-6 receptor antibody by BaF / 6R cells
In order to obtain a cell line exhibiting IL-6-dependent proliferation, a BaF3 cell line expressing human gp130 and human IL-6R was established as shown below. Full-length human IL-6R cDNA was amplified by PCR and cloned into pcDNA3.1 (+) (Invitrogen) to construct hIL-6R / pcDNA3.1 (+). pCOS2Zeo / gp130 is transfected into BaF3 cells by electroporation and selected in the presence of human interleukin-6 (R & D systems) and 100 ng / mL human interleukin-6 soluble receptor (R & D systems) to express human gp130-expressing BaF3 A cell line (hereinafter referred to as BaF / gp130) was established. Furthermore, full-length human IL-6R cDNA was amplified by PCR and cloned into pcDNA3.1 (+) (Invitrogen) to construct hIL-6R / pcDNA3.1 (+). pcDNA3.1 (+) / hIL-6R is introduced into the BaF / gp130 cells prepared above by electroporation and selected in the presence of human interleukin-6 (R & D systems), and human IL-6R-expressing BaF3 cells A stock (hereinafter referred to as BaF / 6R) was established. Since this BaF / 6R grows in the presence of human interleukin-6 (R & D systems), it should be used to evaluate the anti-human IL-6 receptor antibody growth inhibitory activity (ie, human IL-6 receptor neutralizing activity). Is possible.

Using BaF / 6R, the human IL-6 receptor neutralizing activity of the anti-human IL-6 receptor antibody was evaluated. After washing BaF / 6R three times with RPMI1640 medium containing 10% FBS, 20 ng / mL human interleukin-6 (TORAY) (final concentration is 10 ng / mL) to be 2.5-5.0 x 10 ⁴ cells / mL mL) and 10% FBS in RPMI1640 medium, and 50 μL was dispensed into each well of a 96 well-plate (CORNING). Next, the anti-human IL-6 receptor antibody was diluted in RPMI1640 containing 10% FBS, and 50 μL was mixed with each well. WST-8 reagent (Cell Counting Kit-8, Dojindo Laboratories, Inc.) that was cultured for 3 days at 37 ° C and 5% CO ₂ and diluted 2-fold with PBS was added at 20 μL / well. Absorbance at 450 nm (reference wavelength 620 nm) was measured using SUNRISE CLASSIC (TECAN). After culturing for 2 hours, the absorbance at 450 nm (reference wavelength: 620 nm) was measured again, and the human IL-6 receptor neutralizing activity was evaluated using the change in absorbance for 2 to 4 hours as an index.

[Reference Example 5]
Evaluation of binding activity of each mutation-modified antibody by competitive ELISA of anti-human GPC3 antibody The binding activity of the prepared antibody was determined using a competitive ELISA. A soluble GPC3 core polypeptide (SEQ ID NO: 207) prepared to 1 μg / ml was added to a 96-well plate at 100 μl per well. The plate was allowed to stand overnight at 4 ° C., and the soluble GPC3 core polypeptide was immobilized on the plate. The soluble GPC3 core polypeptide immobilized on the plate was washed 3 times with wash buffer using Skan WASHER400 (Molecular Devices), added with 200 μl blocking buffer and blocked at 4 ° C for 30 min or longer. It was done. The plate on which the soluble GPC3 core polypeptide had been immobilized and blocked was then washed 3 times with wash buffer using Skan WASHER400. Thereafter, 200 μl of GPC3-H2L2 antibody with various concentrations or other antibodies and 100 μl of biotinylated GPC3-H2L2 antibody with a final concentration of 0.3 μg / ml were added per well of the plate. Biotinylation of GPC3-H2L2 antibody was performed using Biotin Labeling kit (Roche) according to the kit procedure. The plate was allowed to stand at room temperature for 1 hour, and then washed 5 times with a washing buffer using Skan WASHER400 (Molecular Devices). The plate containing 100 μl of Goat anti streptabidin Alkaline phosphatase (ZYMED) diluted 20,000 times with the substrate buffer per well was allowed to stand at room temperature for 1 hour, and then washed with Skan WASHER400. Washed 5 times. Phosphatase Substrate (Sigma) was prepared to 1 mg / ml using a substrate buffer, and 100 μl per well was added and allowed to stand for 1 hour. Using Benchmark Plus (BIO-RAD), the absorbance at 405 nm of the reaction solution in each well was measured using a control absorbance of 655 nm.

[Reference Example 6]
Method for evaluating affinity of anti-human IL-31 receptor antibody to IL-31 receptor by Biacore
1. Preparation of soluble human IL-31 receptor Using the human IL31 receptor cDNA as a template, only the extracellular region was amplified by the PCR method, and a FLAG tag sequence was added to the C-terminus, which was then incorporated into an expression vector for mammalian cells. . 10 μg of this linearized vector was introduced into Chinese hamster ovary cell line DG44 by electroporation (BioRad Gene PulserII, 25 μF, 1.5 kV) to obtain a cell line exhibiting high expression. A soluble NR10 was obtained by purifying the cell line from a culture supernatant obtained by mass culture using an anti-FLAG antibody column (manufactured by SIGMA) and gel filtration. The amino acid sequence of the soluble human IL31 receptor is shown in SEQ ID NO: 246.

2. Evaluation of affinity for soluble human IL-31 receptor by Biacore
Using Biacore T100 (GE Healthcare Bioscience), kinetic analysis of antigen-antibody reaction between anti-human IL-31 receptor antibody and soluble human IL-31 receptor was performed. Rec-Protein A (ZYMED) (hereinafter referred to as Protein A) is immobilized on the sensor chip by a method known to those skilled in the art, the antibody is captured on this immobilized Protein A, and the antigen is reacted as an analyte to react with the antibody. Antigen interaction was measured. Each antibody was prepared to bind an appropriate amount to Protein A / G. The soluble human IL-31 receptor used as the analyte was prepared at 0, 38.5, 77.0, and 154 nM using HBS-EP +. First, the antibody solution was bound to Protein A / G, the analyte solution was allowed to interact there for 3 minutes, then switched to HBS-EP + and the dissociation phase was measured for 5 minutes. After measurement of the dissociated phase, the sensor chip was regenerated by washing with 10 μL of 10 mM glycine-HCl (pH 1.5). This binding / dissociation / regeneration was defined as one cycle of analysis. Various antibodies were measured according to this cycle. The obtained sensorgrams were analyzed kinetically using Biacore T100 Evaluation Software (GE Healthcare Bioscience), Biacore's exclusive data analysis software.

  According to the present invention, a method for modifying the isoelectric point of an antibody by modifying the charge of at least one amino acid residue that can be exposed on the surface of a complementarity determining region (CDR) region while retaining the binding activity to the antigen, Provided are a method for purifying a multispecific antibody, a method for improving the plasma pharmacokinetics of an antibody, a pharmaceutical composition containing an antibody with an altered isoelectric point as an active ingredient, and a method for producing the same. By modifying the isoelectric point of the antibody, it is possible to efficiently purify the multispecific antibody with high purity. Further, by modifying the isoelectric point of the antibody, it becomes possible to improve the pharmacokinetics in plasma, and it is possible to reduce the frequency of administration and to exert a therapeutic effect continuously. The antibody obtained by the method of the present invention retains the binding activity to the antigen.

Claims (10)

A method for producing a pharmaceutical composition comprising a polypeptide comprising a variable region of an antibody with controlled plasma pharmacokinetics, comprising:
The method comprises modifying the charge of at least one amino acid residue that may be exposed on the surface of a complementarity determining region (CDR) of a polypeptide comprising the variable region of an antibody;
The modification in the charge of an amino acid residue is performed by at least one amino acid substitution selected from the group consisting of the following (i) ~ (iii),
(i) substitution of a charged amino acid with a non-charged amino acid;
(ii) substitution of a charged amino acid with an amino acid having the opposite charge to the amino acid; and
(iii) substitution of a non-charged amino acid with a charged amino acid ;
At least one amino acid residue that can be exposed on the surface of the CDR,
Amino acid residues at positions 31, 62, 64, 65 and 97 by Kabat numbering in the heavy chain variable region or positions 24, 27, 53, 54 and 55 by Kabat numbering in the light chain variable region Two or more amino acid residues selected from amino acid residues, or
Amino acid residues at positions 31, 62, 64 and 65 by Kabat numbering in the heavy chain variable region or amino acid residues at positions 24, 27, 53, 54 and 55 by Kabat numbering in the light chain variable region Two or more amino acid residues selected from
Said method . A method for producing a pharmaceutical composition comprising a polypeptide comprising a variable region of an antibody with controlled plasma pharmacokinetics, comprising:
(a) modifying a nucleic acid encoding a polypeptide containing the amino acid residue such that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide is modified;
(b) culturing the host cell so that the nucleic acid is expressed;
(c) a method comprising recovering a polypeptide comprising an antibody variable region from a host cell culture,
Here, modification of the charge of the amino acid residue is performed by at least one amino acid substitution selected from the group consisting of the following (i) ~ (iii),
(i) substitution of a charged amino acid with a non-charged amino acid;
(ii) substitution of a charged amino acid with an amino acid having the opposite charge to the amino acid; and
(iii) substitution of a non-charged amino acid with a charged amino acid ;
At least one amino acid residue that can be exposed on the surface of the CDR,
Amino acid residues at positions 31, 62, 64, 65 and 97 by Kabat numbering in the heavy chain variable region or positions 24, 27, 53, 54 and 55 by Kabat numbering in the light chain variable region Two or more amino acid residues selected from amino acid residues, or
Amino acid residues at positions 31, 62, 64 and 65 by Kabat numbering in the heavy chain variable region or amino acid residues at positions 24, 27, 53, 54 and 55 by Kabat numbering in the light chain variable region Two or more amino acid residues selected from
Said method .   The method according to claim 1 or 2, wherein the polypeptide comprising the variable region of the antibody further comprises an FcRn binding region.   The method according to any one of claims 1 to 3, wherein the polypeptide containing the variable region of the antibody is an IgG antibody.   The method according to any one of claims 1 to 4, wherein the polypeptide comprising the variable region of the antibody is a chimeric antibody, a humanized antibody or a human antibody. The method according to any one of claims 1 to 5, wherein the charged amino acid is at least one amino acid selected from the group consisting of the following (i) to (v):
(i) glutamic acid (E);
(ii) aspartic acid (D);
(iii) lysine (K);
(iv) arginine (R); and
(v) Histidine (H). The method according to any one of claims 1 to 6 , wherein the charge modification of at least one amino acid residue that can be exposed on the surface of the CDR is a modification that retains the binding activity to the antigen. The method according to any one of claims 1 to 7 , further comprising measuring the isoelectric point value of the modified polypeptide after modifying the charge. The method according to any one of claims 1 to 8 , wherein the polypeptide containing the variable region of the antibody is a multispecific polypeptide that binds to at least two kinds of antigens. The modification in the charge of an amino acid residue is an modification that alters the theoretical isoelectric point 1.0 above, the method according to any one of claims 1-9.

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